Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig‐Maximilians‐Universität München

NOVEL CHEMICAL TOOLS FOR THE

MODULATION OF TWO PORE CHANNEL 2

Susanne Daniela Gerndt aus Schweinfurt

2020

Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Franz Bracher betreut.

Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet.

München, den 29.06.2020

______Susanne Daniela Gerndt

Dissertation eingereicht am: 01.07.2020

1. Gutachter: Prof. Dr. Franz Bracher 2. Gutachter: Prof. Dr. Dr. Christian Grimm

Mündliche Prüfung am 27.07.2020

Danksagung

An dieser Stelle möchte ich mich ganz herzlich bei allen bedanken, die diese Arbeit durch viele große und kleine Beiträge erst ermöglicht haben:

Allen voran Herrn Prof. Dr. Franz Bracher für dieses interessante und abwechslungsreiche Thema und seine stets großzügige Unterstützung und Förderung während der vergangenen Jahre.

Ebenso bedanken möchte ich mich bei den Mitgliedern der Prüfungskommission, vor allem bei Prof. Dr. Dr. Christian Grimm, nicht nur für die freundliche Übernahme des Koreferats, sondern auch für die Möglichkeit mich in ein mir fachfremdes Thema einzuarbeiten und viel Neues zu lernen.

Weiter möchte ich auch meinen Kooperationspartnern danken, sei es in München Frau Prof. Dr. Angelika Vollmar, in Leipzig Prof. Dr. Michael Schaefer oder anderswo.

Bei den Mitarbeitern der Analytikabteilung der Chemie und Pharmazie bedanke ich mich auch recht herzlich für die zahlreichen Messungen und die Hilfe bei analytischen Fragestellungen.

Einen herzlichen Dank richte ich auch an die Arbeitsgruppen Biel, Grimm und Schaefer, die mich im Rahmen gemeinsamer Kooperationen freundlich aufgenommen und unterstützt haben.

Natürlich möchte ich mich auch bei allen aktuellen und ehemaligen Kollegen aus dem Arbeitskreis Bracher für die gute Zusammenarbeit, die Unterstützung und die großartige Zeit bedanken. Vielen Dank für die zahlreichen schönen Stunden mit euch, vielen Dank für die tollen Gespräche und vielen Dank für das gemeinsame Lachen in unserem Relatively Nice Network.

Vielen Dank auch an die Studenten, sei es den Hiwis, den Wahlpflicht- oder Bachelorstudenten, die mich in diesem Projekt unter meiner Betreuung tatkräftig unterstützt haben und so einen wertvollen Beitrag zu dieser Arbeit geleistet haben.

Herzlichsten Dank gilt auch all denen, die sich dazu bereit erklärt haben diese Arbeit Korrektur zu lesen.

Bei meinen Liebsten, meinen Freunden und meiner Familie, möchte ich mich auch für die viele Geduld, Sorge, Anteilnahme, Unterstützung, Trost aber auch die Freude, Zuversicht und Liebe, die mir geschenkt wurde bedanken. Ohne euch wäre das Alles nicht möglich gewesen. Mein größter Dank gilt Tim, der immer an mich glaubt, jeden Tag.

Vielen Dank!

TABLE OF CONTENTS

Table of contents

1 Introduction 1 1.1 Two pore channels 1 1.2 Two pore channels and disease 2 1.2.1 Two pore channels and cancer 3 1.2.2 Two pore channels and viral infections 3 1.3 Modulators of two pore channels 4 1.4 Tetrandrine (1) – opportunities and limitations 7

2 Objectives 9

3 Results and discussion 11 3.1 Identification of novel TPC2 activators 11 3.2 Synthesis of TPC2-A1-N (16) and analogs 14 3.3 Synthesis of TPC2-A1-P (17) and analogs 22 3.4 Pharmacological investigation of TPC2 agonists 31 3.4.1 Confirmation of TPC2-A1-N (16) and TPC2-A1-P (17) as TPC2 agonists 31 3.4.2 Structure-activity relationships for TPC2-A1-N (16) and analogs 33 3.4.3 Structure-activity relationships for TPC2-A1-P (17) and analogs 39 3.4.4 Further results of the novel TPC2 agonists 44 3.4.5 Malleable cation selectivity of TPC2 46 3.4.6 The race to identify new TPC activators 48 3.5 A new generation of TPC2 inhibitors 51 3.5.1 Collection of alkaloids and 1-benzylisoquinolines 51 3.5.2 Synthesis plan 53 3.5.3 Synthesis of a new generation of TPC2 inhibitors 58 3.6 Pharmacological investigation of TPC2 antagonists 71 3.6.1 First identification of the truncated TPC2 inhibitors 71 3.6.2 Analysis of the compound library 74 3.6.3 TPC2 and cancer 78 3.6.4 Separation of the enantiomers 82 3.6.5 Expanding the TPC2 inhibitor panel 83 3.7 Side Projects 85 3.7.1 The isoquinoline-benzylisoquinoline alkaloid rac-muraricine (213) 85 3.7.2 The TRPML inhibitors ML-SI1 (214) and ML-SI3 (215) 86

I TABLE OF CONTENTS

4 Summary 88

5 Experimental part 95 5.1 Preparation of TPC2 activators 97 5.1.1 General procedures 97 5.1.2 Synthetic procedures 98 5.1.2.1 Synthesis of TPC2-A1-N (16) and analogs 98 5.1.2.2 Synthesis of TPC2-A1-P (17) and analogs 148 5.2 Preparation of TPC2 inhibitors 168 5.2.1 General procedures 168 5.2.2 Synthetic procedures 170 5.2.2.1 Synthesis of arylethylamine building blocks 170 5.2.2.2 Synthesis of enol ethers 176 5.2.2.3 N-Acyl Pictet-Spengler reaction 179 5.2.2.4 Lithium alanate reductions of carbamates and deprotections of carbonates 183 5.2.2.5 Chan-Evans-Lam couplings 190 5.2.2.6 Further reactions 201 5.2.2.7 Substances received from other sources 204 5.3 Biological methods 208 5.3.1 High throughput screening (HTS) 208 5.3.2 Single cell calcium imaging 208 5.3.3 MTT 209 5.3.4 Agar diffusion test 210

6 Appendices 211 6.1 Abbreviations 211 6.2 References 214

II INTRODUCTION

1 Introduction

“The most fruitful basis of the discovery of a new drug is to start with an old drug.”

Sir James Black

With these words the pharmacologist and Nobel laureate Sir James Black established a basis for drug repurposing. The fact that an already known drug is used for a new indication that differs from the original indication is called drug repurposing. Thereby new targets can be identified for the old drugs. Another approach, beside these new targets, is that the scaffolds of the old drugs or natural compounds can also serve as an inspiration for the synthesis of novel small-molecules[1].

This thesis deals with two pore channel 2 (TPC2) as the target and tetrandrine (1, Figure 1) as the natural compound with the need to be developed. In 2015, Sakurai et al. linked the entry of Ebola virus into host cells to two pore channels (TPCs). The inhibition of TPCs with tetrandrine (1) halted virus trafficking and prevented infection[2], creating the fundament for this work.

Figure 1: Chemical structure of the alkaloid tetrandrine (1), identified as a TPC2 inhibitor.

1.1 Two pore channels

Two pore channels are part of the family of voltage-gated ion channels and are localized in the endo-lysosomal system. They share parts of the sequential identity with voltage-gated calcium

[3] (Cav) and sodium (Nav) channels . Two isoforms of two pore channels (TPC1 and TPC2) can be found in primates. TPC1 is preferentially found in early endosomal compartments and TPC2 in late endosomal and lysosomal compartments[4-6]. In 2016, Kintzler and Stroud presented the crystal structure of the NAADP antagonist trans-Ned-19 (2) bound to TPC1 from Arabidopsis

1 INTRODUCTION thaliana[7]. The cryo-EM structure of human TPC2 has recently been resolved by She et al.[8] and verifies the dimeric nature and the duplicated domains of two pore channels. Two times six transmembrane helices form one domain and two domains form the ion channel. TPCs are non-selective cation channels, permeable to calcium and sodium and they are involved in endo-lysosomal trafficking, autophagy, mTOR and TFEB signaling[9]. Whether TPC2 is sodium or calcium permeable was subject of controversial debates in the last decade. While TPC2 was first described as non-selective, calcium permeable cation channel activated by NAADP (3)[10-14], other research groups claimed that TPC2 was a sodium-selective channel activated

[15, 16] by the endo-lysosomal phosphoinositide PI(3,5)P2 (4) . Both views independently received support in the past couple of years[5, 17-21] and the controversy has just recently been resolved[22] in the course of this project, as described in chapter 3.4.5.

Two pore channels have been a hot topic in recent literature, not only because of their controversially discussed ion permeability but also because of their involvement in various diseases like viral and bacterial infections and cancer cell migration. This makes two pore channels a promising target for drug research.

1.2 Two pore channels and disease

As mentioned before, in recent years TPCs have emerged as highly exciting potential drug targets for a number of diseases associated with the endo-lysosomal system[23]. Dysfunction of TPCs has been found to interfere with cholesterol trafficking resulting in fatty liver disease[5], the β-adrenergic stimulation of glucagon secretion in diabetes[24] and β-adrenoceptor signaling in the heart in cardiovascular diseases[25]. Melanin production and pigmentation are also influenced by TPC activity[26-28]. Furthermore, Parkinson’s disease caused by LRRK2 mutations has been linked to TPC functions[29]. In addition, several bacterial toxins such as diphtheria toxin, anthrax toxin, cholera toxin, or Pasteurella multocida toxin have been shown to require functional TPCs[4, 6]. Beside its role in bacterial infections, TPCs have been demonstrated to play a role in various infectious diseases such as Ebola filovirus, Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or HIV-1 retrovirus infections [2, 30-33]. Furthermore there is a growing amount of evidence that TPCs are necessary for sustaining cancer hallmarks like cell migration and neoangiogenesis[9, 34-36].

2 INTRODUCTION

1.2.1 Two pore channels and cancer

The role of TPCs in cancer research is complex, because TPCs are involved in different processes during carcinogenesis. The fact that prostate cancer patients with high levels of TPC2 gene expression showed poor survival probabilities was a strong evidence for the correlation between TPCs and cancer hallmarks[37]. Nguyen et al. confirmed these results and showed that silencing of TPC2 reduced migration and adhesion of invasive lung tumor cells[36]. The endo-lysosomal system promotes trafficking of integrins. During cell migration integrins pass the endocytic cycle. They are released from their substrate and taken back by endocytosis. If this recycling process is disturbed, cancer cell migration in vitro and metastasis in vivo are impaired[36]. Vascular endothelial growth factors (VEGF) play an important role during neoangiogenesis, including vascularization of solid tumors. Inhibition of VEGF reduces vessel formation, a fundamental step for the vascularization of solid tumors. Hence VEGF inhibitors are already used to treat cancer[38]. The role of TPC2-mediated calcium release in neoangiogenesis was demonstrated in TPC knockout mice. In TPC2, but not in TPC1, knockout mice the VEGF pathway was inhibited and vessel formation was reduced[35]. Whether TPCs and especially TPC2 are involved in other cancer hallmarks, such as proliferation and dysregulation of the cellular energy metabolism[38], remained largely unknown and was investigated in the studies presented in chapter 3.6.3.

1.2.2 Two pore channels and viral infections

Especially in the year 2020, viral infections are a hot topic. The corona pandemic has hospitals reaching their bed capacities, causing many deaths. The WHO is currently promoting a study for drug repurposing, including drugs that have already been in use for HIV and malaria therapy[39]. The connection between viral infections and two pore channel activity has already been shown for Ebola virus, MERS-CoV, HIV-1 and lately for the new SARS-CoV-2[2, 30-33]. Ou et al. described that SARS-CoV-2 enters the cell via endocytosis and that PIKfyve and TPC2 were required for entering the cells[33]. The enzyme PIKfyve is responsible for phosphorylation of phosphatidylinositol 3-phosphate in C-5 position to PI(3,5)P2 (4), an endogenous compound that is activating TPCs and is regulating endo-lysosomal vesicle trafficking. Inhibition of the mucolipin TRPML1 (see chapter 1.3) using an unfortunately not further specified TRPML1 inhibitor was ineffective, while inhibition of TPC2 with tetrandrine (1) halted the SARS-CoV-2 entry[33]. Tetrandrine (1) has already been shown to successfully prevent Ebola virus infection by blocking TPC1 and TPC2 activity[2]. A link to the transporter protein Niemann-Pick C1 has been demonstrated for SARS-CoV[40], similar to Ebola virus[2]. For Ebola virus both isoforms of TPC, TPC1 and TPC2, are required for virus trafficking and infectivity while for SARS-CoV-2 this correlation is not confirmed. Many facts are still unknown with respect to these correlations

3 INTRODUCTION and further investigation is of high interest. Having tailored tool compounds would surely boost studies to assess the correlation of TPCs and SARS-CoV-2 in more detail and would benefit the combat against viral infections.

1.3 Modulators of two pore channels

As part of the controversial debate about the ion permeability of TPCs, two different hydrophilic activators have been used in the past decade. The phosphoinositide phosphatidylinositol 3,5- bisphosphate (PI(3,5)P2 (4)), a major constituent of endo-lysosomal membranes, has first been described by Dong et al.[41] as an activator of transient receptor potential mucolipin (TRPML) channels. TPCs and TRPMLs share PI(3,5)P2 (4) as activator but are only distantly related to each other in terms of sequence similarities[15, 16]. Both channel families share a number of functional features. They are non-selective cation channels located in endo-lysosomes and permeable to sodium and calcium. Both are involved in endo-lysosomal trafficking, autophagy,

[9] TFEB and mTOR signaling . Thus, TPCs are, in addition to PI(3,5)P2 (4), activated by nicotinic acid adenine dinucleotide phosphate (NAADP (3))[10-14]. Both activators are highly hydrophilic and are not plasma membrane permeable. A plasma membrane permeable variant of NAADP (3), NAADP/AM (5) is commercially available[42, 43]. This lipophilic acetoxymethyl (AM) ester prodrug (5) is, however, due to its instability very limited in use (Figure 2).

Figure 2: Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2 (4)) and NAADP (3) and its membrane permeable variant NAADP/AM (5). Inositol 4 carries phosphate glycerol esters and various fatty acids in C-1 position (e.g. n = 16 or 18).

The same applies to the membrane permeable PI(3,5)P2 variant. Dinkel et al. published a synthesis for the PI(3,5)P2 variant with increased lipophilicity and membrane permeability. AM- esters were introduced to protect phosphate residues, as it was already known from NAADP/AM (5), and the secondary alcohol groups were protected as butyrates[44]. Unfortunately, these derivatives are only available via an extensive, multi-step synthesis.

As there is an urgent need for lipophilic, plasma membrane permeable small-molecule activators of TPCs, a high-throughput screening for the identification of novel, small-molecule

4 INTRODUCTION

TPC2 activators was arranged. The screening of an 80.000 compound strong library obtained from Roche (Basel, CH) was performed within this project.

For the identification of TPC inhibitors, different approaches were pursued. Penny et al. performed a virtual screening of Ebola virus entry inhibitors in order to identify possible TPC blockers[31]. The hits were confirmed as TPC2 inhibitors by blocking NAADP-evoked calcium release in a sea urchin egg homogenate model and via patch clamp experiments. This group identified different dopamine receptor-affine drugs and selective estrogen receptor modulators (SERMs) as additional TPC blockers: the anti-psychotics trifluoperazine, prochlorperazine, thioridazine and fluphenazine (6) as well as the SERMs clomiphene, tamoxifene, toremifene, bazedoxifene and raloxifene (7).

Figure 3: Structures of TPC2 inhibitors. (A) Most promising TPC2 inhibitors identified by Penny et al.[31]: raloxifene (7) and fluphenazine (6). (B) The flavonoid naringenin (8) and trans-Ned-19 (2).

The most promising TPC2 inhibitors identified by Penny et al., raloxifene (7) and fluphenazine

(6), are approved drugs (Figure 3 A). Both exhibited low IC50 values in endo-lysosomal patch

[31] clamp experiments (0.63 µM and 8.2 µM) after stimulation with PI(3,5)P2 (4) . The SERM raloxifene (7) is known to reduce breast cancer risk[45] and is used for treatment and prevention of osteoporosis. Moreover, it is limited in its approval to postmenopausal women regarding the influence upon hormone levels. It has also been reported that raloxifene (7) blocks L-type and T-type voltage-sensitive calcium channels[46, 47] as well as Kv4.3 channels[48]. Fluphenazine (6) is an anti-psychotic drug used to treat psychotic disorders such as schizophrenia with severe adverse effects, in particular extrapyramidal effects including acute dystonia, akathisia, tardive dyskinesia and Parkinsonism[49]. Fluphenazine (6) blocks postsynaptic mesolimbic

5 INTRODUCTION dopaminergic D1 and D2 receptors in the brain, neuronal voltage-gated sodium channels[50] and the ATP-sensitive potassium (KATP) channel[51].

Besides its known broad spectrum of targets in mammalian organisms, the flavonoid naringenin (8, Figure 3 B) was also identified as a TPC2 blocker by Pafumi et al.[52]. Furthermore, it was investigated on its inhibitory effect in cell migration[53]. Naringenin (8) can also block several other ion channels: the melastatin channel TRPM3 (calcium channel)[54],

[55] [56] voltage-gated sodium channels (Navs) and cardiac HERG channels (potassium channel) . Additionally it enhances the activity of large-conductance Ca2+-activated potassium (BK) channels[57]. However, naringenin (8) is most prominent for the inhibitory effect on cytochrome P450 enzymes like CYP1A2[58] or CYP3A4[59] – a property which bears the risk of undesired drug-drug interactions.

The NAADP antagonist and therefore indirect TPC2 blocker trans-Ned-19 (2, Figure 3 B) has been known for many years[60] and the crystal structure solved by Kintzler and Stroud confirmed the interaction with TPCs[7], albeit discussed controversially.

Examining these compounds, it is of interest that many of them are discussed in their relationship to cancer. Aforementioned, raloxifene (7) is known to reduce breast cancer risk[45]. Naringenin (8) was investigated on its inhibitory effect in cell migration[53] and to affect VEGF- evoked tube formation[52] but requires very high concentrations (≥ 500 µM), as well as trans- Ned-19 (2). The latter, trans-Ned-19 (2), was able to reduce NAADP-induced Ca2+ release and prevented the activation of the VEGF signaling pathway[35] as well as cancer cell migration[36] in high micromolar doses (≥ 100 µM). All these compounds are not applicable for in vivo experiments due to the side effects or required high doses. Though, there is an emerging need for the development of efficacious TPC2 inhibitors with drug-like properties and antitumor activity to study the involvement of TPC2 in cancer.

Tetrandrine (1, Figure 3 A) is the most prominent TPC inhibitor in current literature and was first described as such by Sakurai et al. in 2015[2]. It is known to also block voltage-gated Ca2+ channels, large-conductance Ca2+-activated K+ (BK) channels and intracellular Ca2+ pumps[61]. Additionally, tetrandrine (1) was recently identified to effectively inhibit cancer cell migration[36] and infections with Ebola[2], MERS-CoV[30] and SARS-CoV-2[33] viruses. These effects were most likely induced via directly acting on TPCs. Tetrandrine (1) is used in different studies as exemplary TPC inhibitor and therefore represents a promising lead structure for the development of TPC2 blockers.

6 INTRODUCTION

1.4 Tetrandrine (1) – opportunities and limitations

The bisbenzylisoquinoline alkaloid tetrandrine (1) is isolated from the Asian plant Stephania tetrandra and is used in traditional Chinese medicine for treating asthma, tuberculosis, malaria or hyperglycemia[62]. Apart from its traditional applications, tetrandrine (1) is under pharmacological investigation. Its activity spectrum extends inflammations, antidiabetic effects, antimicrobial and anticancer activities, the use as antioxidant, P-gp and calcium channel inhibition[62], as depicted in Figure 4. Plenty of these pharmacological activities can also be related to TPCs, as tetrandrine (1) is a known TPC1 and 2 inhibitor[2]. Studies about viral infections (Ebola[2], MERS-CoV[30] and SARS-CoV-2[33]) and the influence of TPC2 were carried out using tetrandrine (1) but it is also under investigation for bacterial infections (e.g. Staphylococcus aureus)[63] or fungus (e.g. Candida albicans)[64]. Also for investigations in cancer research connected with TPCs, tetrandrine (1) is state-of-the-art[36]. Tetrandrine (1) was also highlighted in a study to prevent diabetes[65], which now can be related to its inhibitory effect on TPCs[24].

Figure 4: Overview of the activity spectrum of tetrandrine (1).

P-glycoprotein (P-gp) however, appears as novel target for tetrandrine (1), which was identified as a highly potent P-gp inhibitor[66, 67]. P-gp is an efflux pump with a broad substrate spectrum. These transporters pump foreign substances out of cells and are therefore responsible for multidrug resistance of tumors. Correlations to TPCs have not been investigated yet.

Despite all these positive features tetrandrine (1) also has a few drawbacks. One example is the poor solubility. In some studies the sample preparation was not commented[68, 69] and others reported that they needed to acidify the injection solution for dissolution before in vivo application[2, 70]. These circumstances make tetrandrine (1) poorly applicable for in vivo studies.

7 INTRODUCTION

Furthermore, a high toxicity is described in animal models. There are hints that the toxicity of tetrandrine (1) is based on oxidative metabolism by cytochromes P450s 3A4 and 3A5. The authors claimed that the methoxy group at C-12 (Figure 4) is metabolized to a free phenol and further oxidized to give a reactive para-quinone methide intermediate that can directly be trapped by bio-nucleophiles like glutathione[70, 71]. This toxicity is based on the reaction with other bio-nucleophiles like cysteine residues in proteins that would then be destroyed, while glutathione would detox the para-quinone methide intermediate. The broad activity spectrum of tetrandrine (1) is maybe related to its ability to inhibit TPC1 and 2, but other side effects are certain to occur and need to be reduced as well.

The alkaloid is mainly obtained by extraction of the roots of Stephania tetrandra or by complex, multi-step chemical synthesis. The synthesis of enantiomerically pure tetrandrine (1) involves more than 20 steps[72]. A recently published synthesis from our research group shortened the sequence to 12 steps after which the racemic mixture of tetrandrine (1) is obtained[73].

The opportunities and limitations of tetrandrine (1) generate a suitable basis for the development of this long known bioactive compound. The outlined drawbacks were aimed to be overcome in this project by simplification of the structure, substitutions and derivatizations.

8 OBJECTIVES

2 Objectives

TPCs are of rising importance. Involved in many diseases but mechanistically poorly understood, there is an emerging need for further investigations. Despite its drawbacks, tetrandrine (1) was the most promising candidate for developing new TPC inhibitors.

New anti-cancer agents with the potential to block TPCs were e.g. easily accessible derivatives of tetrandrine (1) with modifications in C-5[74, 75] and C-14[76, 77] position, other related natural compounds like fangchinoline (9, also originated from Stephania tetrandra)[78], analogs of berbamine (10, from Berberis amurensis)[79], or seco-derivatives as dauricine (11, from Menispermum dauricum DC)[80] and neferine (12, from Nelumbo nucifera)[81], as depicted in Figure 5.

Figure 5: Structures of the alkaloids fangchinoline (9), berbamine (10), dauricine (11) and neferine (12). Berbamine analogs are glycosides in C-12 position of berbamine (10). Differences to tetrandrine (1) are marked in red.

All these molecules, however, lacked variability because they were obtained from natural extracts and their derivatization was restricted to certain functional groups. If isolation from plants was not possible or a diversified substitution pattern was encouraged, complex multi- step syntheses need to be performed. Iturriaga-Vásquez et al. designed simplified and more accessible congeners of tetrandrine (1), only representing one benzylisoquinoline half of the molecule and carrying extensions like O-benzyl groups (Figure 6). These derivatives had

[82] similar potential to block L-type calcium channels (Cavs) like tetrandrine (1) . The fact that

TPCs were a family intermediate of channels related to voltage-gated calcium (Cav) and

[83] sodium channels (Nav) suggested that these simplified structures could have high potential to block TPCs and further have high anticancer or antiviral activity. In addition, these truncated variants could overcome the lack of bioavailability, increase efficacy, be less toxic, more

9 OBJECTIVES specific, have a good solubility and are easily accessible and variable via a short, fast and efficient synthesis.

Figure 6: Truncation of tetrandrine (1) should give monomers, resembling one half of tetrandrine (1)[82].

To analyze TPC inhibitors in cellular systems the unique lysosomal patch clamp technique is required. Channel inhibition can be observed by this technique right after activation with the endogenous TPC2 activators PI(3,5)P2 (4) or NAADP (3). Though this is a complex technique and only a few people in the world are able to perform these complex experiments. In order to analyze large compound libraries of potential TPC2 blockers, an easily accessible experimental setup was crucial. Ca2+ imaging experiments, using different Ca2+ indicating dyes like Fura-2 (13) or Fluo-4 (14) are well known but require intact cells and cell-permeable activators.

Therefore, novel small-molecule activators of TPC2 need to be identified first, synthesized and further analyzed to investigate their biological and pharmacological properties. These activators would then be used in a second step as chemical tools for the identification and analysis of TPC2 blockers, but in addition may play an independent role as lead structures for potential therapeutics in lysosomal storage diseases (LSDs).

10 RESULTS AND DISCUSSION

3 Results and discussion

The results of this thesis are presented in this section and are divided into two main parts, agonists and antagonists of TPC2.

Starting with the performance of a high-throughput screening, the first part represents the identification of novel TPC2 activators. These hits were confirmed by synthesis, characterization and subsequent pharmacological experiments. Furthermore, analogs of these hits were synthesized to analyze structure-activity relationships (SAR). This was achieved by single cell Ca2+ imaging experiments. Concentration-effect relationships were analyzed and further results from cooperation partners are briefly described to present of the whole outcome of this interdisciplinary project.

The second main part of this chapter describes the synthesis and characterization of truncated tetrandrine analogs as novel TPC2 inhibitors. A Ca2+ imaging protocol to analyze TPC2 inhibitors, using our novel TPC2 activators, was developed. Biological activity of the inhibitors is of high interest and the effects on cancer were evaluated by cooperation partners. Furthermore, the enantiomers of the most promising substances were separated to distinguish between eutomer and distomer.

Additionally, results of some side projects are presented and briefly described.

3.1 Identification of novel TPC2 activators

Lysosomal storage diseases (LSDs) are a very hot topic not only in academic research, but also in industrial research. A cooperative project with F. Hoffmann-La Roche (Basel, CH) provided the opportunity to use two of their compound libraries (Xplore X30 and X50) to perform a high-throughput screening (HTS). The aim of this screening was to identify novel activators of Battenin (CLN3) and TPC2.

CLN3 is a putative lysosomal transporter or channel of unknown function but with a high relevance in inherited neurodegenerative disorder (Batten disease). Mutations in CLN3 lead to the loss of CLN3 function, causing Batten disease, also known as juvenile neuronal ceroid lipofuscinosis (JNCL) disease[84, 85]. Batten disease is the most common NCL with an onset of symptoms in childhood, characterized by progressive loss of vision, seizures, psychomotor disturbances and eventually premature death[86, 87]. As CLN3 and TPC2 are both lysosomal transmembrane proteins both proteins were screened in parallel serving as controls for each other.

In cooperation with Dr. Phuong Nguyen from Prof. Dr. Angelika Vollmar’s group (LMU, Munich) and Prof. Dr. Michael Schaefer’s group (Rudolf Boehm Institute, Leipzig) the high-throughput

11 RESULTS AND DISCUSSION screening was performed in Leipzig, using a custom-made fluorescence imaging plate reader (FLIPR) built into a robotic liquid handling station (for 96 tips), as previously described[88]. HEK293 cells stably expressing the plasma membrane variant of hTPC2, TPC2L11A/L12A-RFP and another cell line stably expressing the plasma membrane variant of hCLN3, CLN3L253A/I254A -RFP were used[22]. For the screening around 80.000 compounds on 221 384- well plates from two different libraries (X50 and X30) were provided by Roche (Basel, CH). All compounds were tested on both cell lines and double positive hits were excluded to ensure the accuracy of the hits in a first instance.

The membrane permeable, single-wavelength fluorescent dye Fluo-4/AM (15) was selected as Ca2+-indicator. With this indicator experiments in cell suspension were possible, which was ideal for high-throughput screenings (Figure 7 A). Fluo-4/AM (15) entered the cells as lipophilic ester and was hydrolyzed inside the cells by esterases. Resulting Fluo-4 (14) is barely fluorescent, but fluorescence increases at least 100 times on Ca2+ binding when excited at 488 nm (Figure 7 B). An increase in fluorescence reflects rise in cytoplasmic Ca2+ levels caused by activation of the ion channels[89, 90].

Figure 7: The principle of Fluo-4 based Ca2+-imaging. (A) Short schematic overview of Fluo-4 based Ca2+-imaging. Fluo-4/AM (15) can enter the cell as a membrane permeable molecule. Esterases cleave the AM esters and the free dye 14 remains. Fluo-4 (14) can complex Ca2+ ions which results in increased fluorescence at 516 nm. (B) Structures of Fluo-4/AM (15) and the free Fluo-4 (14) anion.

Compounds were screened on both cell lines. Hitlists of single hits were generated giving a total amount of 118 CLN3 primary hits and 133 TPC2 primary hits (Figure 8 A). Since

12 RESULTS AND DISCUSSION availability of the compounds as well as the quantity was limited, only the strongest hits (highest fluorescence increase) for each cell line were selected and further investigated by concentration-effect experiments. Thus, only 25 CLN3 primary hits and 12 TPC2 primary hits were further considered. Concentration-effect experiments in the range of 50 µM to 24 nM resulted in two secondary CLN3 hits (A09 and C15) and four secondary TPC2 hits (N19, N10, H07 and L13). These secondary hits were all evaluated in Fura-2 based Ca2+-imaging experiments (see chapter 3.4 for the method). None of the analyzed CLN3 hits showed a specific activation. One TPC2 hit had no effect and another one showed unspecific or toxic effects. Hence only two final hits for TPC2 (N19, named TPC2-A1-N (16) and H07, named TPC2-A1-P (17)) remained that were able to activate the TPC2 ion channel (Figure 8 B). The activity of these two compounds was further confirmed by the electrophysiological endo- lysosomal patch clamp technique.

Figure 8: Summary of the high-throughput screening. (A) Simplified overview for the stepwise identification of the two TPC2 screening hits. (B) Structure of the two screening hits N19 (new name: TPC2-A1-N (16)) and H07 (new name: TPC2-A1-P (17)), as presented to us by Roche (Basel, CH).

To verify the postulated structure and assess SAR, generally applicable syntheses for both hits had to be developed and the resulting compounds had to be re-evaluated via Fura-2 calcium imaging

13 RESULTS AND DISCUSSION

3.2 Synthesis of TPC2-A1-N (16) and analogs

Starting with TPC2-A1-N (16), first a synthesis to confirm the structure of the hit had to be developed. A fast, efficient and versatile synthesis was desired. Thus, a compound library can be generated to examine SAR. Variations of both aromatic rings, the necessity of a secondary amide, as well as the extension of the middle part were investigated. Sjogren et al. published a two-step synthesis for related α-cyano-β-hydroxypropenamides, which resulted in exactly the desired scaffold of TPC2-A1-N (I, Scheme 1)[91].

Scheme 1: Retrosynthetic overview showing the synthesis of α-cyano-β-hydroxypropenamides (I) in two steps. By using different aniline (IV) and benzoic acid (III) building blocks, a large variability of combinations is possible.

Following these procedures an C-acylation using different activated benzoic acid building blocks (III, Scheme 1) and NaH as strong base should easily yield desired hydroxypropeneamides (I, Scheme 1). Therefore the required α-cyano amide (II, Scheme 1) could be synthesized using different aniline building blocks (IV, Scheme 1), 2-cyanoacetic acid (18) and a standard coupling reagent as DCC. This synthesis resulted in the desired α-cyano-β-hydroxypropenamides (I, Scheme 1) in only two steps and without need for purification by column chromatography, which gave the opportunity to establish a broad compound library.

Scheme 2: Synthesis of 2-cyano-N-(4-(trifluoromethyl)phenyl)acetamide (SGA-34, 19), according to Sjogren et al.[91].

Starting with the structure confirmation of the hit TPC2-A1-N (16) commercially available 4-(trifluoromethyl)aniline (20), 2-cyanoacetic acid (18) and DCC were used in DMF. After one hour the reaction was completed. Purification by filtration and recrystallization yielded the pure 2-cyano-N-(4-(trifluoromethyl)phenyl)acetamide (SGA-34, 19) in high yield (73%, Scheme 2).

14 RESULTS AND DISCUSSION

Only a few modifications in equivalents were made compared to the original literature in order to achieve the best results. For the synthesis of variably substituted α-cyano amides this method was applied (II, Table 1). This resulted in mainly p-substituted α-cyano amides (entry 1-14, Table 1), as well as unsymmetrically (entry 15-19, Table 1) and symmetrically substituted aryls (entry 20, 21, Table 1). Electron-withdrawing groups like halide, nitro, nitrile or carbonyl substituents represented the majority. The methyl group (entry 2, Table 1) was introduced as the electron-donating bioisostere to the screening hit’s trifluoromethyl group (entry 1, Table 1). A variant with a tertiary amide (entry 22, Table 1) and another one being derived from a benzylamine (entry 23, Table 1) completed the α-cyano amides. Yields were all moderate to high, very poor only for N-benzyl-acetamide (entry 23, Table 1).

Table 1: Overview of the results for the synthesis of α-cyano amides (II). Different aniline building blocks (IV and a homologous benzylamine in entry 23) and 2-cyanoacetic acid (18) were used as starting materials.

entry starting material (IV) product (II) yield 2-cyano-N-(4-(trifluoromethyl)phenyl)acetamide (SGA- 1 R = 4-CF3 (20) 73% 34, 19)

2 R = 4-CH3 2-cyano-N-(p-tolyl)acetamide (21) 71% 3 R = H 2-cyano-N-phenylacetamide (22) 76%

4 R = 4-OCH3 2-cyano-N-(4-methoxyphenyl)acetamide (23) 41% 5 R = 4-Cl N-(4-chlorophenyl)-2-cyanoacetamide (24) 77% 6 R = 4-Br N-(4-bromophenyl)-2-cyanoacetamide (25) 40% 7 R = 4-F 2-cyano-N-(4-fluorophenyl)acetamide (26) 75% 8 R = 4-I 2-cyano-N-(4-iodophenyl)acetamide (27) 77%

9 R = 4-NO2 2-cyano-N-(4-nitrophenyl)acetamide (28) 62% 10 R = 4-CN 2-cyano-N-(4-cyanophenyl)acetamide (29) 78% 11 R = 4-Ac N-(4-acetylphenyl)-2-cyanoacetamide (30) 54% 12 R = 4-OPr 2-cyano-N-(4-propoxyphenyl)acetamide (31) 62%

13 R = 4-OCF3 2-cyano-N-(4-(trifluoromethoxy)phenyl)acetamide (32) 72%

14 R = 4-COOCH3 methyl 4-(2-cyanoacetamido)benzoate (33) 65% 15 R = 2-Br, 4-Cl N-(2-bromo-4-chlorophenyl)-2-cyanoacetamide (34) 69% 16 R = 2-I 2-cyano-N-(2-iodophenyl)acetamide (35) 70%

17 R = 2,4-F2, 3-Cl N-(3-chloro-2,4-difluorophenyl)-2-cyanoacetamide (36) 68%

18 R = 3,4-(OCH3)2 2-cyano-N-(3,4-dimethoxyphenyl)acetamide (37) 76%

19 R = 2,3-Cl2 2-cyano-N-(2,3-dichlorophenyl)acetamide (38) 29%

20 R = 2,6-Br2 2-cyano-N-(2,6-dibromophenyl)acetamide (39) 40% N-(3,5-bis(trifluoromethyl)phenyl)-2-cyanoacetamide 21 R = 3,5-(CF3)2 81% (40)

15 RESULTS AND DISCUSSION

N-methyl-4- 2-cyano-N-methyl-N-(4- 22 74% (trifluoromethyl)aniline (trifluoromethyl)phenyl)acetamide (41) 4-(trifluoromethyl) 23 2-cyano-N-(4-(trifluoromethyl)benzyl)acetamide (42) 7% benzylamine

For the synthesis of TPC2-A1-N (16) acylation of the appropriate α-cyano acetamide (SGA- 34, 19) was performed using 3,5-dichlorobenzoyl chloride (43) and NaH in THF[91]. After one hour the reaction was completed and purification by filtration and recrystallization yielded pure 2-cyano-3-(3,5-dichlorophenyl)-3-hydroxy-N-(4-(trifluoromethyl)phenyl)acrylamide (TPC2-A1- N, 16) in high yield (69%, Scheme 3). NMR analysis of TPC2-A1-N (16) showed that the enol- form is favored compared with the keto-form, as described below.

Scheme 3: Synthesis of 2-cyano-3-(3,5-dichlorophenyl)-3-hydroxy-N-(4-(trifluoromethyl)phenyl)- acrylamide (TPC2-A1-N, 16), following a procedure published by Sjogren et al.[91].

This procedure was beneficial in many ways. It was fast and the easy work up gave highly pure crystalline products. These products did not need purification by FCC, which saved time and material. Furthermore FCC would also cause problems, as it is reported that 1,3-diketones form chelate complexes with metal ions, e.g. Fe3+[91-93]. The commonly used silica in chemical laboratories contains slight impurities of Fe3+, which would result in colorful contaminated products. Different α-cyano amides (II, Table 2) were already prepared and plenty benzoic acid building blocks (III) were commercially available. Depending on biological results a variety of compounds could be synthesized by different combinations of these building blocks. Hence, a set of α-cyano-β-hydroxypropenamides (I, Table 2) was synthesized using this method. This resulted in a compound library of 44 TPC2-A1-N analogs. The newly synthesized α-cyano amides (blue, II, Table 2) were used for different moieties on the anilide side (blue, entry 1-7, 10, 11, 14, 15, 18-20, 27-32, 43, Table 2) of the molecule. Widely varying benzoic acid building blocks (green, III, Table 2) were commercially available, which gave the opportunity to introduce 17 different aryl groups (green, entry 1, 8, 9, 12, 13, 17, 21-23, 34, 36- 39, 41, 42, 44, Table 2). Combinations of different amide (II, Table 2) and benzoic acid (III, Table 2) building blocks resulted in 6 more compounds (entry 16, 24-26, 33, 35, Table 2). One variant with a tertiary amide (entry 40, Table 2) and another one replacing the anilide moiety with a benzyl amide (entry 45, Table 2), completed the compound list. Nearly all yields are

16 RESULTS AND DISCUSSION moderate to high, which indicates that this protocol is extendable to a wide variety of α-cyano amides (II, Table 2) and benzoic acid building blocks (III, Table 2).

Table 2: Overview of the results for the synthesis of α-cyano-β-hydroxypropenamides (I). Different benzoic acid building blocks (III) are combined with the α-cyano amides (II). *If the appropriate acid chloride was not commercially available, the benzoic acid was converted into the acid chloride using thionyl chloride.

benzoic acid entry amide (II) product (I) yield chloride* (III)

R = 4-CF3 1 R = 3,5-Cl2 (43) 69% (SGA-34, 19)

2 R = H (22) R = 3,5-Cl2 (43) 68%

3 R = 4-Cl (24) R = 3,5-Cl2 (43) 70%

4 R = 4-Br (25) R = 3,5-Cl2 (43) 70%

5 R = 4-CH3 (21) R = 3,5-Cl2 (43) 53%

6 R = 4-F (26) R = 3,5-Cl2 (43) 63%

7 R = 4-I (27) R = 3,5-Cl2 (43) 61%

R = 4-CF3 8 R = H 56% (SGA-34, 19)

R = 4-CF3 9 R = 3,5-(NO2)2 29% (SGA-34, 19)

10 R = 4-OCH3 (23) R = 3,5-Cl2 (43) 61%

17 RESULTS AND DISCUSSION

11 R = 4-NO2 (28) R = 3,5-Cl2 (43) 55%

R = 4-CF3 12 R = 4-NO2 69% (SGA-34, 19)

R = 4-CF3 13 R = 4-Cl 61% (SGA-34, 19)

14 R = 2-Br, 4-Cl (34) R = 3,5-Cl2 (43) 74%

15 R = 3,4-(OCH3)2 (37) R = 3,5-Cl2 (43) 51%

1-methylpyrrole-2- 16 R = H (22) 75% carbonyl chloride

R = 4-CF3 1-methylpyrrole-2- 17 66% (SGA-34, 19) carbonyl chloride

18 R = 2,4-F2, 3-Cl (36) R = 3,5-Cl2 (43) 72%

19 R = 4-CN (29) R = 3,5-Cl2 (43) 38%

20 R = 2-I (35) R = 3,5-Cl2 (43) 52%

R = 4-CF3 21 R = 2,3,4,5,6-F5 56% (SGA-34, 19)

R = 4-CF3 22 R* = 3,5-Br2 42% (SGA-34, 19)

R = 4-CF3 23 R = 2,4,6-Cl3 36% (SGA-34, 19)

18 RESULTS AND DISCUSSION

24 R = 4-Br (25) R = 2,4,6-Cl3 51%

25 R = 2,4-F2, 3-Cl (36) R = 2,4,6-Cl3 50%

26 R = 4-Br (25) R* = 3,5-Br2 44%

27 R = 4-Ac (30) R = 3,5-Cl2 (43) 79%

28 R = 2,6-Br2 (39) R = 3,5-Cl2 (43) 13%

29 R = 2,3-Cl2 (38) R = 3,5-Cl2 (43) 86%

30 R = 4-OPr (31) R = 3,5-Cl2 (43) 63%

31 R = 3,5-(CF3)2 (40) R = 3,5-Cl2 (43) 68%

32 R = 4-OCF3 (32) R = 3,5-Cl2 (43) 83%

33 R = 2,4-F2, 3-Cl (36) R* = 3,5-Br2 76%

R = 4-CF3 34 acetyl chloride 76% (SGA-34, 19)

35 R = 4-CH3 (21) R = 3,5-(CF3)2 50%

R = 4-CF3 36 R = 3,5-(CF3)2 76% (SGA-34, 19)

19 RESULTS AND DISCUSSION

R = 4-CF3 37 R = 3,5-(CH3)2 49% (SGA-34, 19)

R = 4-CF3 38 R = 3,5-(OCH3)2 79% (SGA-34, 19)

R = 4-CF3 39 R = 4-OCF3 48% (SGA-34, 19)

40 N-methyl-acetamide (41) R = 3,5-Cl2 (43) 48%

R = 4-CF3 41 nicotinoyl chloride 51% (SGA-34, 19)

R = 4-CF3 42 R* = 3-Br, 5-I 62% (SGA-34, 19)

43 R = 4-COOCH3 (33) R = 3,5-Cl2 (43) 85%

R = 4-CF3 5-chloronicotinic 44 61% (SGA-34, 19) acid*

45 benzyl-acetamide (42) R = 3,5-Cl2 (43) 55%

The methyl ester group of SGA-133 (85, entry 43, Table 2) could easily be converted into the free carboxylic acid using alkaline hydrolysis. With an excess of LiOH, the ester was cleaved within 3 h at room temperature. Precipitation gave the free acid SGA-137 (88) in high yield (Scheme 4).

Scheme 4: Deprotection of ester SGA-133 (85) using alkaline conditions gave the free acid SGA-137 (88).

20 RESULTS AND DISCUSSION

Sjogren et al. applied a second method to generate α-cyano-β-hydroxypropenamides (I, Table 2). This procedure was using benzoic acid building blocks (III, Table 2) and acetonitrile to give 3-oxopropanenitriles and condensed these with isocyanates or isothiocyanates[91]. More precisely the thioamide analog of TPC2-A1-N (16) was synthesized this way within an Erasmus project, as depicted in Scheme 5. Benzoyl chloride 43 together with the lithium salt of acetonitrile gave the 3-oxopropanenitrile 89. Subsequent condensation with arylisothiocyanate 90 in the presence of NaH yielded SGA-167 (91) in moderate yields, which was in accordance with literature[91]. Due to a complex work-up of the intermediate 89, yields were not as high as on the first route.

Scheme 5: Synthesis of SGA-167 (91) according to Sjogren et al.[91].

Structural analysis of TPC2-A1-N (16) and analogs was a more difficult process as expected. 1H-NMR analysis showed mainly aromatic signals and 13C NMR spectra of the numerous fluorinated products showed plenty carbon fluorine splits. The carbon fluorine coupling resulted in 1J coupling constants of around 270 Hz, 2J coupling constants of around 30 Hz, 3J coupling constants of around 3.5 Hz and a multiplicity of n + 1. The 1J coupling constant was observed in every 13C spectrum of a fluorine containing substance, the more distant ones were only observed in the absence of heteroatoms. As known from related 1,3-dicarbonyl compounds such as acetylacetone, two tautomeric structures are possible, the keto- and the enol-form, which could also be observed by 1H-NMR. It is reported, that the enol-form of acetylacetone is more stable than the keto-form, due to the formation of a hydrogen bond between one carbonyl and the enol-hydroxy group[94]. For β-ketoamides a hydrogen bond between the β-ketone group and the amide NH is also possible (“keto-amide” form; Scheme 6). Both forms were found by Laurella et al. in studies of tautomeric equilibria of β-ketoamides. Furthermore these authors discovered that electron-withdrawing groups at the benzoyl ring stabilize the enol- amide tautomer, electron donors stabilize the keto-amide form and bulky groups in C-2 position favor the enol-form as well[95]. Transferred to the scaffold of TPC2-A1-N (16), the enol-tautomer was more likely to be the fitting style, as the nitrile residue is highly electronegative. This was further confirmed by NMR analysis. In most 1H-NMR spectra no standard or shifted hydroxyl group was observed, but nevertheless no proton in C-2 position was found. In addition, this carbon was always indicated as quaternary in 13C/HSQC analysis.

21 RESULTS AND DISCUSSION

Scheme 6: Keto-enol tautomerism of the blank TPC2-A1-N scaffold. The two most likely tautomers to occur are displayed, the keto-amide (left) and the enol-amide (right). Both structures are able to form a hydrogen bond to stabilize the conformation.

Collectively, 47 compounds were synthesized in moderate to high yields, including the hit TPC2-A1-N (16), the amide SGA-34 (19) as half of the molecule, two drugs (prinomide (58) and teriflunomide (76)) and a large variety of analogs. All of them were fully characterized by NMR, TLC, HRMS, IR and melting points and the purities were confirmed by analytical HPLC.

3.3 Synthesis of TPC2-A1-P (17) and analogs

The same procedure as for TPC2-A1-N (16) needed to be applied for TPC2-A1-P (17). In more detail, the hit TPC2-A1-P (17) needed to be confirmed, a synthesis to generate TPC2-A1-P (17) and analogs had to be developed and a variety of analogs had to be synthesized and characterized for structure-activity analysis.

Examining the structure of TPC2-A1-P (17) the highly substituted pyrrole moiety stands out. Pyrrole syntheses have been known for decades and syntheses of highly substituted pyrroles were thoroughly examined by many groups. Nevertheless, construction of the appropriately substituted pyrrole ring was the key step. The free carboxylic acid (I, Scheme 7) could be generated by alkaline deprotection of an ester intermediate (II, Scheme 7), which is generated by suitable pyrrole synthesis. The easily available and variable α-halogenated acetophenone (IV, Scheme 7), β-ketoester (V, Scheme 7) and primary amine (III, Scheme 7) building blocks are the basis for pyrrole formation (Scheme 7).

Scheme 7: Retrosynthetic overview for the synthesis of TPC2-A1-P (17) and analogs (I) derived from different α-halogenated acetophenone (IV), β-ketoester (V) and primary amine (III) building blocks.

22 RESULTS AND DISCUSSION

The Hantzsch pyrrole synthesis is one famous reaction to generate pyrroles[96]. Condensation of primary amine (III, Scheme 8) and β-ketoester (V, Scheme 8) building blocks gives an enamino ester intermediate (VI, Scheme 8). C-Alkylation by the α-halogenated acetophenone (IV, Scheme 8) gives a 2-substituted enamino ester (VII, Scheme 8) which undergoes intramolecular cyclocondensation to give the desired 2,3,5-trisubstituted pyrrole (II, Scheme 8)[96-98].

Scheme 8: Commonly accepted mechanism for the Hantzsch pyrrole synthesis, yielding a multi- substituted pyrrole (II).

Zhao et al. published a microwave-assisted one-pot method for the synthesis of N-substituted 2-methyl-1H-pyrrole-3-carboxylate derivatives[99]. This method is notable because of the absence of solvents and catalysts as well as the fast and easy implementation. The appropriate α-halogenated acetophenone to yield TPC2-A1-P (17) is not commercially available. Therefore, two commercially available α-bromo acetophenones (92 and 93) were used for model reactions. Hereby, building block 93 would provide a product very similar to TPC2-A1- P (17), with only the trifluoromethoxy group being replaced by a methoxy group. Together with β-ketoester (94) and cyclohexanemethanamine (95) they were added into one microwave reactor each. After irradiation at 8 bar and 240 °C for 1 h, no residues of bromoacetophenones 92 and 93 were detected. Pyrrole 96 was isolated in low yield, though no product formation could be observed for the pyrrole 97 closely related to TPC2-A1-P (17) (Scheme 9). Hence this method was not further pursued.

Scheme 9: Synthetic approach for the Hantzsch pyrrole synthesis to yield the methoxy analog of TPC2- A1-P 97 and ester 96.

23 RESULTS AND DISCUSSION

Another method, related to the Hantzsch synthesis, is the Paal-Knorr pyrrole synthesis[100, 101]. Condensation of 1,4-diketone building blocks (VIII, Scheme 10) with a primary amine building block (III, Scheme 10) under protic or Lewis acidic conditions yields multi-substituted pyrroles (II, Scheme 10)[102]. The drawback of this method is, that the 1,4-diketone building blocks (VIII, Scheme 10) have to be synthesized in an additional step. A procedure, published by Kang et al.[103], exactly describes this route. With α-halogenated acetophenone (IV, Scheme 10) and β-ketoester (V, Scheme 10) building blocks 1,4-diketones (VIII, Scheme 10) are synthesized using NaH as base. The authors used ammonium acetate in acetic acid to yield N-unsubstituted pyrroles, or primary amine building blocks (III, Scheme 10) with p- toluenesulfonic acid in ethanol to receive N-alkyl pyrroles[103]. Most primary amine building blocks (III, Scheme 10) that will be used are liquids, therefore they don’t need organic solvents to be dissolved and acetic acid is chosen as preferred acid.

Scheme 10: General reaction equation for the formation of 1,4-diketone building blocks (VIII)[103] and subsequent Paal-Knorr pyrrole synthesis yielding a highly substituted pyrrole derivative (II)[102].

In model reactions α-bromoacetophenones 92 and 93 together with β-ketoester 94, KI and NaH gave the 1,4-diketones 98 and 99. After standard work-up these diketones (98 and 99) were pure enough for further proceedings and following Paal-Knorr reaction using cyclohexanemethanamine (95) yielded pyrroles SGA-43 (96) and SGA-54 (97) in high yields over two steps. KI was added for an in situ Finkelstein reaction to boost the conversion of the first step. For the sake of time and material savings the 1,4-diketone building blocks (VIII, Scheme 10) were used right away after preparation, without further purification or analysis.

Scheme 11: Model reactions for the synthesis of 2,3,5-trisubstituted pyrroles, yielding SGA-43 (96) and SGA-54 (97).

24 RESULTS AND DISCUSSION

The final step in the synthesis of the desired carboxylic acids was the deprotection of the esters. Esters can be cleaved by hydrolysis in acidic or alkaline conditions. Kang et al. described a method using NaOH[103], which is the reason why alkaline conditions were chosen. For screening of deprotection conditions ester SGA-43 (96) was chosen. Using NaOH in dioxane/H2O at room temperature only gave starting material after 15 h (entry 1, Table 3).

[104] Changing to LiOH, THF/H2O and reflux gave the same result (entry 2, Table 3), as well as

[105] refluxing with NaOH in dioxane/H2O (entry 3, Table 3). A pressure tube was used to increase the power of the reaction, but still no deprotection was observed (entry 4, Table 3).

To generate maximum power, a microwave reaction was applied. With dioxane/H2O rough conditions could be achieved and after 2 h total conversion of the ester 96 was observed (entry 5, Table 3). For better handling LiOH was examined, also giving the carboxylic acid 100 in high yield (entry 6, Table 3).

Table 3: Screening of conditions for the ester saponification using SGA-43 (96).

entry base solvent temperature / conditions time product

1 NaOH dioxane/H2O (5:1) rt 15 h only s.m.

2 LiOH THF/H2O (4:1) 60 °C / reflux condenser 12 h only s.m.

3 NaOH dioxane/H2O (5:1) 100 °C / reflux condenser 15 h only s.m.

4 NaOH dioxane/H2O (5:1) 100 °C / pressure tube 18 h only s.m.

microwave reactor (pmax = 8 bar, 5 NaOH dioxane/H2O (5:1) 2 h 93% Pmax = 80 W, Tmax = 160 °C)

microwave reactor (pmax = 8 bar, 6 LiOH dioxane/H2O (5:1) 2 h 89% Pmax = 200 W, Tmax = 180 °C)

With this newly developed method for the synthesis of carboxylic acid (I, Scheme 10), only the access to the starting materials needed to be examined. Most starting materials are commercially available and just two α-halogenated acetophenones (IV, Scheme 10) had to be synthesized to give all designed analogs for complete structure-activity relationships: 2- Bromo-1-(2-(trifluoro-methoxy)phenyl)ethan-1-one (101) and 1-(5-bromo-2-(trifluoromethoxy)- phenyl)-2-chloroethan-1-one (102).

Inspired by a poorly detailed patented procedure[106] mono-bromination of 2'-(trifluoromethoxy)- acetophenone (103) was carried out under acidic conditions. Only one equivalent of N-

25 RESULTS AND DISCUSSION bromosuccinimide (NBS) and a catalytic amount of p-toluenesulfonic acid were used to yield the desired bromoketone 101 in moderate yield (Scheme 12).

Scheme 12: Mono-bromination of acetophenone 103 in acidic conditions to give bromoketone 101.

For the synthesis of the hit TPC2-A1-P (17), chloroketone 102 needed to be synthesized. Schlosser et al. published a procedure for lithiation of 1-bromo-4-(trifluoromethoxy)benzene (104) with LDA at C-3 (directed metalation), followed by carboxylation at -100 °C with dry ice[107]. The authors claimed that these conditions reduce the competing ortho- or meta- lithiation (positions to the OCF3 group) in a ratio of 99:1 to receive a regioselective ortho- product. As the desired ortho-lithiation is favored, the reaction was carried out at -78 °C using the Weinreb amide 2-chloro-N-methoxy-N-methylacetamide (105) instead of dry ice. Though the desired α-chloroketone 102 did not occur and only decomposition of the starting material was observed (a, Scheme 13). Same applied to the use of freshly prepared LDA, according to Schlosser et al. (b, Scheme 13)[107]. Furthermore a Friedel-Crafts acylation was performed, using AlCl3 and chloroacetyl chloride in 1,2-dichloroethane (EDC), but no product formation was observed (c, Scheme 13).

Scheme 13: Attempted chloroacetylation of 104. Different reaction conditions were screened. a) 104, LDA, THF, -78 °C, 4 h; then 2-chloro-N-methoxy-N-methylacetamide (105) in THF, -78 °C, 5 min. b) 104, LDA, generated out of DIPA and n-BuLi, -78 °C, 2 h; then 2-chloro-N-methoxy-N-methylacetamide (105) in THF, -78 °C, 5 min. c) 104, AlCl3, chloroacetyl chloride, EDC, 0 °C – 50 °C, 5 h.

Lui et al. developed a method for the oxidative trifluoromethylation of phenols[108]. Among their examples there were ortho- and para-halogenated phenols, which seemed promising. The reaction was performed with commercially available phenol 106 in a custom made glove box, but again no product could be isolated (Scheme 14).

26 RESULTS AND DISCUSSION

Scheme 14: Reaction conditions for the attempted trifluoromethylation of phenol 106.

Another attempt to synthesize chloroketone 102 involved an iodine-lithium exchange, which should be favored over the bromo-lithium exchange[109]. Weinreb amide 105 was used to give the ketone 102 in order to avoid side reactions and over-additions. According to Nahm and Weinreb, a stable chelate complex is formed as intermediate, which avoids further additions to the ketone. Aqueous acidic work up then could yield the desired ketone (Scheme 15 A)[110]. The reaction was performed using commercially available iodine 108 and n-BuLi at -78 °C. After 20 minutes, Weinreb amide 105 was added and the reaction was monitored by TLC and NMR. The NMR sample was taken from the reaction mixture and after mini work-up dried under nitrogen flow. 1H-NMR analysis clearly showed total conversion of the starting material and formation of a new substance with all NMR signals required for the desired ketone 102 after 1 h. After standard work-up of the whole reaction and NMR analysis of the material, no product could be observed anymore. Therefore, it was assumed that the desired ketone 102 was highly volatile and not even stable under reduced pressure, as a rotary evaporator generates (40 °C, 1 mbar). The newly developed work-up avoided rotary evaporation. The product was dried overnight under a nitrogen flow and gave ketone 102 in moderate yield (Scheme 15 B).

Scheme 15: Synthesis of α-chloroketone 102 using in situ generated organolithium reagents and Weinreb amide 105. (A) Reaction mechanism according to M. Weinreb[110]. (B) Successful reaction conditions for the synthesis of α-chloroketone 102.

27 RESULTS AND DISCUSSION

With all building blocks at hand and a well-established route, the synthesis of TPC2-A1-P (17) and analogs could be applied. Finally ester SGA-140 (109, entry 1, Table 4) could be synthesized in moderate yield using α-chloroketone 102, ethyl acetoacetate (94) and cyclohexanemethanamine (95). Deprotection of ester 109 gave the hit compound TPC2-A1-P (17) in good yield. Varying the α-halogenated ketone building block (IV, Table 4) allowed the synthesis of seven more esters that were all isolated in moderate to high yields (entry 2-7, 13, Table 4). The bioisosteric ester SGA-54 (97) was lacking the trifluoromethoxy group at C-2 position of the aromatic ring and was instead bearing a methoxy group (entry 3, Table 4). The 2,5-dichloro variant SGA-48 (110) also shared the substitution pattern of the hit compound (entry 4, Table 4). Esters SGA-59 (111) and SGA-61 (112) both had para-substituents (entry 5, 6, Table 4) and SGA-62 (113) was the 2,4-difluoro analog (entry 7, Table 4). Ester 114 was lacking the bromine in C-5 position of the aromatic ring (entry 13, Table 4). All of these esters were further deprotected to yield the carboxylic acids SGA-50 (100), SGA-55 (115), SGA-52 (116), SGA-66 (117), SGA-67 (118), SGA-68 (119) and SGA-162 (120) in moderate to high yields (entry 2-7, 13, Table 4). These compounds had in common that their α-halogenated ketone building blocks (IV, Table 4) were stable, which is the reason why they could be easily purified. Simple FCC yielded the pure esters. Purification attempts of esters synthesized with the volatile α-chloroketone 102 were very complicated and time consuming. A first set of biological experiments revealed that the ester SGA-140 (109) does in comparison to the carboxylic acid TPC2-A1-P (17) not show any effect in Ca2+ imaging experiments (Figure 15). Therefore there was no need to purify and isolate the pure esters anymore and FCC was just performed as a pre-cleaning process. This was of great importance, because the precipitating of the carboxylic acids was otherwise not successful and additional extractions had to be included. The absence of colorful impurities was critical as this would disturb experiments using fluorescent dyes.

Table 4: Products synthesized according to the newly elaborated method for the synthesis of highly substituted pyrrolecarboxylic acids (I). *cy = cyclohexyl

entry R1 R R2 ester (II) acid (I)

R = 2-OCF3, 1 R = CH3 R = CH2cy* 5-Br

46% 54%

28 RESULTS AND DISCUSSION

2 - R = CH3 R = CH2cy*

53% 89%

R = 2-OCH3, 3 R = CH3 R = CH2cy* 5-Br

95% 63%

4 R = 2,5-Cl2 R = CH3 R = CH2cy*

46% 81%

5 R = 4-F R = CH3 R = CH2cy*

97% 88%

6 R = 4-OCH3 R = CH3 R = CH2cy*

51% 90%

7 R = 2,4-F2 R = CH3 R = CH2cy*

78% 88%

R = 2-OCF3, 8 R = CH3 R = pentyl not isolated 5-Br

15% overall yield

R = 2-OCF3, 9 R = CH3 R = benzyl not isolated 5-Br

10% overall yield

29 RESULTS AND DISCUSSION

R = 2-OCF3, R = 10 R = CH2cy* not isolated 5-Br CH2CH3

9% overall yield

R = 2-OCF3, 11 R = CH3 R = iPr not isolated 5-Br

12% overall yield

R = 2-OCF3, 12 R = phenyl R = CH2cy* not isolated 5-Br

28% overall yield

13 R = 2-OCF3 R = CH3 R = CH2cy*

95% 93%

Different β-ketoester building blocks (V, Table 4) were used to generate SGA-152 (121) and SGA-154 (122), both with larger residues in C-2 position of the pyrrole (entry 10, 12, Table 4). Modifications in 1-position of the pyrrole were achieved with altering primary amine building blocks (III, Table 4) to give carboxylic acids SGA-149 (123) with a long and SGA-153 (124) with a branched side chain as well as SGA-150 (125) with a benzyl moiety (entry 8, 9, 11, Table 4). All yields are low to moderate but over two steps acceptable.

Collectively, a synthesis for TPC2-A1-P (17) was developed that was also suitable for synthesizing analogs. In total 13 esters were synthesized and 8 of them isolated, including the ester SGA-140 (109). Alkaline deprotection under microwave irradiation yielded the hit 17 itself and 12 analogs varying all substituents of the pyrrole moiety. These 21 substances were fully characterized by NMR, TLC, HRMS, IR and melting points and the purity was confirmed by analytical HPLC.

30 RESULTS AND DISCUSSION

3.4 Pharmacological investigation of TPC2 agonists

The agonistic effects of the synthesized screening hits TPC2-A1-N (16) and TPC2-A1-P (17) and their analogs were confirmed by Fura-2 single cell calcium imaging and SAR were analyzed. Concentration-effect relationships using Fluo-4 calcium imaging were analyzed and further results from cooperation partners are briefly described for presentation of the whole outcome of this interdisciplinary project. Finally our novel TPC2 agonists were compared to activators, recently published by Zhang et al.[111]. All studies were performed in close cooperation with Prof. Dr. Michael Schaefer’s group (HTS, performed by myself, Fluo-4 based experiments, performed by Nicole Urban), Prof. Dr. Dr. Christian Grimm’s as well as Prof. Dr. Martin Biel’s groups (Fura-2 based calcium imaging, performed by myself and patch clamp experiments, performed by Dr. Yu-Kai Chao and Dr. Cheng-Chang Chen).

3.4.1 Confirmation of TPC2-A1-N (16) and TPC2-A1-P (17) as TPC2 agonists

Single cell Ca2+ imaging experiments had confirmed the two HTS hits, using HEK293 cells transiently expressing the plasma membrane variant of human TPC2 (hTPC2L11A/L12A)[22]. The membrane permeable, ratiometric dye Fura-2/AM (126) was selected as Ca2+ indicator for these experiments. Ca2+ imaging using Fura-2 (13) enables the detection of changes in calcium levels in adherent cells[112].

Figure 9: The principle of Fura-2 based Ca2+-imaging. (A) Schematic overview of Fura-2/AM based Ca2+-imaging. Fura-2/AM (126) can enter the cell as membrane permeable AM ester. Esterases cleave the AM esters and the free dye 13 remains. Excitation wavelength for Fura-2 (13) changes upon complexation from 380 nm (no Ca2+) to 340 nm (with Ca2+). Emission can be detected at 510 nm. (B) Structures of Fura-2/AM (126) and the free Fura-2 (13) anion.

31 RESULTS AND DISCUSSION

Fura-2/AM (126) can enter cells as a lipophilic AM ester and is hydrolyzed inside the cells by esterases. The Fura-2 (13) anion can complex Ca2+ ions and shifts its absorption maximum from 380 nm to 340 nm, both emitting at 510 nm (Figure 9 A and B). While Ca2+ concentrations rise, an increase of fluorescence for excitation at 340 nm can be observed. The level of transfection, as well as the loading of the cell with Fura-2 (13) have an impact on the fluorescence intensity. To avoid measurement inaccuracies, the ratio of chelated Fura-2 (340 nm) divided by free Fura-2 (13, 380 nm) is determined.

Figure 10: Confirmation of TPC2-A1-N (16) and TPC2-A1-P (17) as TPC2 agonists. (A) Representative Ca2+ signals recorded from HEK293 cells loaded with the ratiometric Ca2+ indicator, Fura-2 (13) and stimulated with TPC2-A1-N (16, 10 µM) and monitored for 400 s. Cells were transiently transfected with plasma membrane targeted human TPC2 (hTPC2L11A/L12A). Highlighted lines represent the mean response from a population of cells. Shaded traces represent responses of single cells (n = 10 cells for hTPC2; n = 3 cells for NT cells). (B) Similar to A except that cells were stimulated with TPC2-A1-P (17, 10 µM) and monitored for 600 s. Shaded traces represent single cells responses (n = 10 cells for hTPC2; n = 3 cells for NT cells). (C) Similar to A except that cells were stimulated with TPC2-A1-P (17, 30 µM). Shaded traces represent single cells responses (n = 9 cells for hTPC2; n = 3 cells for NT cells). (D)

32 RESULTS AND DISCUSSION

Statistical analysis of the maximal change in Fura-2 (13) ratio (mean ± SEM) with the number of independent transfections in parentheses. Experiments were performed over the period of 9 months and on the same days, each. ***p < 0.001, using one-way ANOVA followed by Tukey’s post hoc test. (E-F) Representative FLIPR-generated Ca2+ signals (Fluo-4 (14)) in TPC2L11A/L12A–expressing cells (red and blue line) or non-transfected (NT) control cells (black) after addition of TPC2-A1-N (16, E) or TPC2- A1-P (17, F). Experiments were performed by Nicole Urban. (G-H) Concentration-effect relationships for Ca2+ increases (Fluo-4 (14)) in response to different concentrations of TPC2-A1-N (16, G) and TPC2- A1-P (17, H). Experiments were performed by Nicole Urban. (I-J) Representative Ca2+ signals recorded from HEK293 cells loaded with the ratiometric Ca2+ indicator, Fura-2 (13) and sequentially stimulated with TPC2-A1-N (16, 10 µM, I, n = 6 cells) or TPC2-A1-P (17, 30 µM, J, n = 6 cells) and the TRPML agonist ML-SA1 (10 µM). Cells were transiently transfected with human TRPML1∆NC. Means are highlighted and single cell responses are shaded. (K-L) Experiment as in I-J, but HEK293 cells transiently transfected with human TRPML2, or TRPML3. Cells were sequentially stimulated with TPC2- A1-N (16, 10 µM, K, n = 4 cells) or TPC2-A1-P (17, 30 µM, L, n = 4 cells) and the TRPML agonist ML-SA1 (10 µM) or the TRPML2 selective agonist ML2-SA1 (10 µM). Means are highlighted and single cell responses are shaded. Fura-2 based calcium imaging experiments were performed on a Polychrome IV mono-chromator (TILL photonics).

Both screening hits, investigated as the independently synthesized substances, reproducibly evoked Ca2+ signals. Thereby the correct structures of the HTS hits were confirmed. The signals evoked by the compounds showed different kinetics whereby the TPC2-A1-N (16) response reached its plateau faster than TPC2-A1-P response (17, Figure 10 A-B and E-F). The slower activation could be reduced by stimulation with higher concentrations of TPC2-A1- P (17), while control cells still remained unaffected (Figure 10 C). Hence, recommended experimental concentration for TPC2-A1-P (17) is 30 µM. Long term statistical analysis of TPC2-A1-N (16) and TPC2-A1-P (17) showed robust and significant activation of TPC2

(Figure 10 D). Concentration-effect relationships indicated EC50 values of 7.8 μM and 10.5 μM, for TPC2-A1-N (16) and TPC2-A1-P (17), respectively (Figure 10 G and H). Both agonists failed to evoke Ca2+ signals in cells expressing human TRPML1 re-routed to the plasma membrane (TRPML1∆NC)[113, 114] at the recommended working concentrations (Figure 10 I and J). Similar negative results were obtained with the TRPML agonists ML-SA1 and ML2-SA1[115] as positive controls in cells expressing TRPML2 or TRPML3 (Figure 10 K and L). These results indicate that both hits are selective TPC2 agonists with different modes of activation.

3.4.2 Structure-activity relationships for TPC2-A1-N (16) and analogs

Fura-2 based calcium imaging experiments were performed with transiently transfected HEK293 cells, which allowed selection of transfected cells and non-transfected (NT) control cells in one experiment. False positive analogs could straight away be eliminated and unspecific effects on intact cells could be determined. If a compound showed no effect in Ca2+ imaging experiments, a control had to be added. At the beginning of the screening, TPC2-A1- N (16) was not fully established as TPC2 activator and therefore ionomycin (4 µM) was used as a control for a well-functioning experiment. Ionomycin is a membrane permeable Ca2+

33 RESULTS AND DISCUSSION ionophore and causes an intracellular calcium rise by transporting Ca2+ across biological membranes[116, 117]. This is not selective for TPC2, thus ionomycin is a control for the accuracy of the experiment. Stimulation of the cells with a selective activator determines whether a compound is inactive or an inhibitor.

Figure 11: Fura-2 based Ca2+ imaging results for all TPC2-A1-N (16) analogs. (A-B) Fura-2 based Ca2+ imaging results showing the effect of TPC2-A1-N (16) and its analogs (10 μM, each) on HEK293 cells transiently transfected with hTPC2L11A/L12A-YFP (A, red) and control cells (B, grey). The red arrow indicates unspecific effect on control cells, the orange arrow indicates auto fluorescence and the green arrow indicates no effect. Mean values normalized to basal (400 s after compound application) ± SEM of at least three independent experiments with 3–10 cells each are shown. All experiments were performed on a Polychrome IV mono-chromator (TILL photonics).

All synthesized analogs of TPC2-A1-N (16) were tested on their ability to activate TPC2. Out of 46 analogs, 23 substances have shown significant agonistic effects (Figure 11 A) and 6 more barely any activation (SGA-1 (44), SGA-12 (52), SGA-16 (55), SGA-38 (61), SGA-72 (66), SGA-112 (79), Figure 11 A). Ionomycin or TPC2-A1-N (16) were added for the purpose of verifying the accuracy of the experiments. In all experiments with inactive compounds TPC2- A1-N (16) was added at least once. Thus there were not enough data for statistical analysis to verify that the inactive compounds do not inhibit the activation of TPC2. SGA-13 (53) showed increased levels of activation on control cells that could be identified as auto fluorescence

34 RESULTS AND DISCUSSION

(Figure 11 B). Three substances (SGA-27 (56), SGA-85 (73), SGA-132 (84)) also had slight unspecific effects on control cells, that were not related to auto fluorescence. Seven substances (SGA-33 (60), SGA-85 (73), SGA-86 (74), SGA-90 (75), SGA-108 (77), SGA-111 (78), SGA-132 (84)) had comparable efficacy to TPC2-A1-N (16), while all others were less effective.

Analyzing the structures of the 8 most potent TPC2 activators within this series showed that TPC2-A1-N analogs have meta-disubstitution patterns with electron-withdrawing groups located on the benzoyl ring system (Figure 12). In contrast, substitution pattern at the phenyl ring on the anilide side of the molecule was more variable. Electron-withdrawing and -releasing groups (TPC2-A1-N (16), SGA-86 (74) vs. SGA-108 (77)) did not cause significant changes in activity. Substitution patterns could also differ from para to meta (TPC2-A1-N (16), SGA-85 (73)) or even more strongly (SGA-33 (60), SGA-90 (75)).

Figure 12: Structures of the 8 most potent TPC2 activators within the TPC2-A1-N (16) series. Structural differences are marked in magenta.

Harsh changes on the benzoyl moiety (replacement by methyl or pyrrole residues), as demonstrated for the approved drugs teriflunomide (SGA-94, 76) and prinomide (SGA-31, 58) as well as the 4-trifluoromethyl variant of prinomide (SGA-32, 59) led to a complete loss of activity (Figure 13). Teriflunomide (76) and SGA-32 (59) did not show inhibitory effects after activation with TPC2-A1-N (16, Figure 13 B and D). Stimulation with ionomycin (Figure 13 C) indicates the accuracy of the experiment by strong, unspecific calcium influx as described above. Teriflunomide (76) is an approved drug for the treatment of multiple sclerosis and prinomide (58) for rheumatoid arthritis[118, 119]. Dysregulation of calcium homeostasis can be associated with several neurodegenerative disorders like multiple sclerosis (MS)[120], but there is no direct link between MS and TPC2 up to now.

35 RESULTS AND DISCUSSION

Figure 13: Approved drugs structurally related to TPC2-A1-N (16) show no effect on TPC2. (A) Structures of the approved drugs teriflunomide (SGA-94, 76) and prinomide (SGA-31, 58) and analog SGA-32 (59). (B-D) Representative calcium signals from HEK293 cells transiently transfected with hTPC2L11A/L12A-YFP. Cells were stimulated with teriflunomide (76, 10 µM, A, n = 12 cells for hTPC2; n = 7 cells for NT cells), prinomide (58, 10 µM, B, n = 10 cells for hTPC2; n = 6 cells for NT cells) or SGA- 32 (59, 10 µM, C, n = 12 cells for hTPC2; n = 4 cells for NT cells) for 400 s each, followed by activation with TPC2-A1-N (16, 10 µM, B and D, 400 s) or ionomycin (C, 4 µM, 200 s). Highligted lines represent the mean response from a population of cells. Shaded traces represent responses of single cells. Fura- 2 based calcium imaging experiments were performed on a Polychrome IV mono-chromator (TILL photonics).

Thio-analog SGA-167 (91) was synthesized as last compound within an Erasmus project (Figure 14 A). At that time, a new Ca2+-imaging setup was put into operation. Direct comparison from experiments using the old setup (Polychrome IV mono-chromator, TILL photonics) with the new setup (Leica DMi8 live cell microscope) is not possible. Intensities can vary due to the power of the light sources and analysis of the experiments using the manufacturers’ software (TILLvisION or LAS X) could also cause differences in intensities. Therefore, TPC2-A1-N (16) was re-evaluated and used as an established activator to analyze differences in agonistic effects compared to SGA-167 (91). The thio-analog 91 activates TPC2 significantly less than TPC2-A1-N (16, Figure 14 B), clearly indicating very special SAR in this chemotype.

Figure 14: Thio-analog SGA-167 (91) activates TPC2 less than the hit. (A) Structure of SGA-167 (91). Differences to TPC2-A1-N (16) are marked in red. (B) Fura-2 based Ca2+ imaging results showing the

36 RESULTS AND DISCUSSION effect of TPC2-A1-N (16, 10 µM) and its analog SGA-167 (91, 10 μM) on HEK293 cells stably expressing hTPC2L11A/L12A-RFP. Mean values normalized to basal (400 s after compound application) ± SEM of at least three independent experiments with 3–10 cells each are shown. ***p < 0.001 using unpaired student’s t-test. Fura-2 based calcium imaging experiments were performed on a Leica DMi8 live cell microscope.

All findings were affirmed by full analysis of concentration-effect relationships, performed by our cooperation partner Nicole Urban, Rudolf-Boehm-Institute, Leipzig. The Fluo-4 based calcium imaging experiments were chosen because a large number of compounds at different concentrations can be analyzed in a high-throughput manner. The barely activating compounds show either high EC50 values (entry 3, 11, 13, 19, Table 5) or were not calculable because activation started at concentrations above 50 µM. With EC50 values above 100 µM substances were not suitable anymore because such high concentrations can cause unwanted side effects and solubility problems. Substances that were inactive in Fura-2 calcium imaging, did not show agonistic effects in Fluo-4 imaging as well. This confirmed all results from the

Fura-2 imaging experiments. Nearly all EC50 values of the 8 most potent substances are in the same range, thus SGA-33 (60, 23 µM) is higher to some extent (Table 5). SGA-85 (73) and

SGA-132 (84) have a slightly lower EC50 (3.3 µM and 5.3 µM) than TPC2-A1-N (16, 7.8 µM), which is relativized by the fact that control cells showed increased levels of activation in Fura- 2 imaging for SGA-85 (73) and SGA-132 (84, Figure 11 B).

Further analysis of structural motifs shows that replacing the para-trifluoromethyl group on the anilide side of the molecule (R1; blue) gives more opportunities than changes on the benzoyl residue. Electron-withdrawing groups in para-position on the anilide side causes no significant changes (e.g. entry 2, 3, 5, 6, Table 5). Even the introduction of electron-releasing groups in para-position is tolerated to some extent (SGA-4 (47), SGA-84 (72), SGA-108 (77)). For the substitution pattern of the benzoyl ring system (R2, green), meta-disubstitution patterns with electron-withdrawing groups are most beneficial.

Table 5: Structure variations and EC50 values of TPC2-A1-N (16) and the active analogs. No significantly increased efficacies or potencies were observed for the analogs. EC50 (Fluo-4 (14)) values are received 2+ [22] via Fluo-4 based Ca imaging experiments, performed by Nicole Urban as previously described . IC50 values (MTT) are received from in-house MTT experiments, performed by Martina Stadler. *experiment was performed at a later time. n.a. = not applicable.

O OH R1 N R2 H CN IC entry name R1 = R2 = EC = 50 50 (MTT) =

1 TPC2-A1-N (16) 4-CF3 3,5-Cl2 7.8 µM > 50 µM

2 SGA-34 (19) 4-CF3 - n.a. > 50 µM

3 SGA-1 (44) H 3,5-Cl2 35 µM > 50 µM

37 RESULTS AND DISCUSSION

4 SGA-2 (45) 4-Cl 3,5-Cl2 24 µM > 50 µM

5 SGA-3 (46) 4-Br 3,5-Cl2 20 µM > 50 µM

6 SGA-4 (47) 4-CH3 3,5-Cl2 15 µM > 50 µM

7 SGA-8 (48) 4-F 3,5-Cl2 22 µM > 50 µM

8 SGA-9 (49) 4-I 3,5-Cl2 34 µM 48 µM

9 SGA-10 (50) 4-CF3 H n.a. > 50 µM

10 SGA-11 (51) 4-CF3 3,5-(NO2) 2 38 µM > 50 µM

11 SGA-12 (52) 4-OCH3 3,5-Cl2 33 µM > 50 µM

12 SGA-15 (54) 4-CF3 4-NO2 > 100 µM > 50 µM

13 SGA-16 (55) 4-CF3 4-Cl 47 µM > 50 µM

14 SGA-27 (56) 2-Br, 4-Cl 3,5-Cl2 10 µM > 50 µM

15 SGA-28 (57) 3,4-(OCH3)2 3,5-Cl2 n.a. > 50 µM 16 SGA-31 (58) prinomide n.a. > 50 µM 2-cyano-3-(1-methyl-1H-pyrrol-2-yl)-3- 17 SGA-32 (59) hydroxy-N-(4- n.a. > 50 µM (trifluoromethyl)phenyl)acrylamide

18 SGA-33 (60) 2,4-F2, 3-Cl 3,5-Cl2 23 µM > 50 µM

19 SGA-38 (61) 4-CN 3,5-Cl2 51 µM > 50 µM

20 SGA-39 (62) 2-I 3,5-Cl2 > 100 µM > 50 µM

21 SGA-40 (63) 4-CF3 2,3,4,5,6-F5 41 µM > 50 µM

22 SGA-70 (64) 4-CF3 3,5-Br2 13 µM > 50 µM

23 SGA-71 (65) 4-CF3 2,4,6-Cl3 40 µM > 50 µM

24 SGA-72 (66) 4-Br 2,4,6-Cl3 > 100 µM > 50 µM

25 SGA-73 (67) 2,4-F2, 3-Cl 2,4,6-Cl3 n.a. > 50 µM

26 SGA-75 (68) 4-Br 3,5-Br2 22 µM > 50 µM

27 SGA-76 (69) 4-Ac 3,5-Cl2 > 100 µM > 50 µM

28 SGA-77 (70) 2,6-Br2 3,5-Cl2 n.a. > 50 µM

29 SGA-78 (71) 2,3-Cl2 3,5-Cl2 n.a. > 50 µM

30 SGA-84 (72) 4-OC3H7 3,5-Cl2 14 µM > 50 µM

31 SGA-85 (73) 3,5-(CF3)2 3,5-Cl2 3.0 µM 29 µM

32 SGA-86 (74) 4-OCF3 3,5-Cl2 9.5 µM 49 µM

33 SGA-90 (75) 2,4-F2, 3-Cl 3,5-Br2 12 µM > 50 µM 34 SGA-94 (76) teriflunomide n.a. > 50 µM

35 SGA-108 (77) 4-CH3 3,5-(CF3)2 7.1 µM > 50 µM

36 SGA-111 (78) 4-CF3 3,5-(CF3)2 6.2 µM > 50 µM

37 SGA-112 (79) 4-CF3 3,5-(CH3)2 > 100 µM > 50 µM

38 SGA-113 (80) 4-CF3 3,5-(OCH3)2 n.a. > 50 µM

39 SGA-114 (81) 4-CF3 4-OCF3 > 100 µM > 50 µM 2-cyano-3-(3,5-dichlorophenyl)-3-hydroxy-N- 40 SGA-115 (82) methyl-N-(4-(trifluoromethyl)phenyl)- > 100 µM 35 µM acrylamide 2-cyano-3-hydroxy-3-(pyridin-3-yl)-N-(4- 41 SGA-127 (83) n.a. > 50 µM (trifluoromethyl)phenyl)acrylamide

42 SGA-132 (84) 4-CF3 3-Br, 5-I 5.3 µM 32 µM

38 RESULTS AND DISCUSSION

43 SGA-133 (85) 4-COOCH3 3,5-Cl2 21 µM > 50 µM 3-(5-chloropyridin-3-yl)-2-cyano-3-hydroxy-N- 44 SGA-136 (86) n.a. > 50 µM (4-(trifluoromethyl)phenyl)acrylamide

45 SGA-137 (88) 4-COOH 3,5-Cl2 n.a. > 50 µM 2-cyano-3-(3,5-dichlorophenyl)-3-hydroxy-N- 46 SGA-138 (87) > 100 µM 48 µM (4-(trifluoromethyl)benzyl)acrylamide 2-cyano-3-(3,5-dichlorophenyl)-3-hydroxy-N- 47* SGA-167 (91) (4-(trifluoromethyl)phenyl)prop-2- 31 µM > 50 µM enethioamide

The toxicity of all substances was examined by in-house MTT assays using HL-60 cells and was performed by Martina Stadler. The MTT assay indicates the metabolic cell activity and

[121] reflects cell viability . IC50 represents the concentration that is required for half maximal inhibition and compounds with values smaller than 50 µM are acknowledged as cytotoxic. Nearly all analogs and the hit itself did not show toxicity and only 6 substances had slight cytotoxic effects (entry 8, 31, 32, 40, 42, 46, Table 5). Out of the 8 most potent substances, only three substances showed slight toxicity (SGA-85 (73), SGA-86 (74), SGA-132 (84), entry 31-32, 42, Table 5) while the hit compound TPC2-A1-N (16) itself showed no toxicity (entry 1, Table 5).

The HTS hit TPC2-A1-N (16) itself and some of its analogs bearing residues in para-position at the benzoyl residue are known as anthelmintic agents[91]. The authors performed mechanism of action studies using Ascaris mitochondria and have shown that these compounds are uncouplers of oxidative phosphorylation, comparable to salicylanilide anthelmintic agents[122]. There are no indications in current literature that this effect is related to TPC2.

In summary, none of the 46 modified versions of TPC2-A1-N (16, auto-fluorescent SGA-13 (53) already excluded) showed significantly increased efficacies or potencies on TPC2 (Figure 11, Table 5) and the TPC2-A1-N chemotype showed a very flat structure-activity relationship on TPC2. TPC2-A1-N (16) thus remained a promising chemical tool for studies of TPC2.

3.4.3 Structure-activity relationships for TPC2-A1-P (17) and analogs

Fura-2 based calcium imaging experiments were performed as described above for TPC2-A1- N (16) and analogs (Figure 15 A). Experiments with inactive substances were subsequently activated with TPC2-A1-N (16) or stimulated with ionomycin (Figure 15, E and F). Also, for TPC2-A1-P (17) and analogs full concentration-effect experiments were performed by Nicole Urban (Leipzig), using the Fluo-4 based high-throughput setup as described before (Table 6).

39 RESULTS AND DISCUSSION

Similar to the results obtained for TPC2-A1-N (16), modified versions of TPC2-A1-P (17) showed no improvement of potency or efficacy. Virtually all 19 analogs were inactive, only compound SGA-150 (125), in which the N-cyclohexylmethyl residue is replaced by an N-benzyl group (of identical size) showed noteworthy, but reduced activity (Figure 15 A, Table 6). Analysis of SAR revealed that the free carboxylic acid is essential for the activating effect, as the ester SGA-140 (109) was not active (entry 2, Table 6). It may serve as a prodrug of TPC2- A1-P (17) in living systems, but this has not been investigated further. Both, the trifluoromethoxy and the bromine substituent at the phenyl ring were essential for activating TPC2, as exemplified by the inactive methoxy (SGA-55, 115, entry 8, Table 6) and des-bromo (SGA-162, 120, entry 20, Table 6) analogs. Even moderate expansions of the size of the substituent at C-2 position of the pyrrole (methyl in TPC2-A1-P (17) vs. ethyl in SGA-152 (121) and phenyl analog SGA-154 (122), entry 1 vs. 17, 19, Table 6), had the same detrimental effect.

Figure 15. Fura-2 based Ca2+ imaging results for all TPC2-A1-P (17) analogs. (A-B) Fura-2 based Ca2+ imaging results showing the effect of TPC2-A1-P (17) and its analogs (10 µM; each) on HEK293 cells transiently transfected with hTPC2L11A/L12A-YFP (A, blue) and control cells (B, grey). Mean values normalized to basal (400 s after compound application) ± SEM of at least three independent experiments with 3–10 cells each are shown. TPC2-A1-P (17) is highlighted and the analogs are shaded. (C-D) Representative calcium signals from HEK293 cells transiently transfected with hTPC2L11A/L12A-YFP.

40 RESULTS AND DISCUSSION

Cells were stimulated with TPC2-A1-P (17, C, 10 µM, 600 s, n = 10 cells for hTPC2; n = 3 cells for NT cells) or SGA-150 (125, D, 10 µM, 800 s, n = 10 cells for hTPC2; n = 3 cells for NT cells). Highligted lines represent the mean response from a population of cells. Shaded traces represent responses of single cells. Each structure is depicted on the right of the graph and structural differences to TPC2-A1- P (17) are marked in red. (E-F) Experiment as in C-D, but cells were sequentially stimulated with SGA- 50 (100, E, 10 µM) or SGA-149 (123, F, 10 µM) and the TPC2 activator TPC2-A1-N (16, 10 µM) for 400 s each. All experiments were performed on a Polychrome IV mono-chromator (TILL photonics).

Aforementioned, the only fairly tolerable structure modification was the replacement of the cyclohexylmethyl moiety in 1-positon of the pyrrole ring by a benzyl residue (SGA-150, 125, entry 16, Table 6). The efficacy of TPC2-A1-P (17) was significantly higher than of SGA-150 (125) (Figure 15 A) while for both compounds control cells remained unaffected (Figure 15 B). Linear or branched alkyl chains (SGA-149 (123), SGA-153 (124), entry 15, 18, Table 6), however, induced loss of activity.

Fura-2 based single cell calcium imaging traces for the activation of TPC2 were shown in Figure 15 C-F. These experiments showed the slow and weak activation of TPC2 with SGA- 150 (125). This activation was even slower than the abovementioned activation using TPC2- A1-P at 10 µM (17, Figure 15 C and D). This indicated a lower affinity of SGA-150 (125) on TPC2 compared to TPC2-A1-P (17). Exemplary Fura-2 traces for the inactive compounds SGA-50 (100) and SGA-149 (123) were shown in Figure 15 E and F. Both substances had no effect on TPC2 and following activation using TPC2-A1-N (16) was successful, indicating that SGA-50 (100) and SGA-149 (123) were neither activators, nor inhibitors.

Table 6: Structure variations and EC50/IC50 values of TPC2-A1-P (17), its ester SGA-140 (109) and all 2+ other analogs. EC50 values were received via Fluo-4 based Ca imaging experiments, performed by [22] Nicole Urban (Leipzig) as previously described . IC50 values (MTT) were received from in-house MTT experiments, performed by Martina Stadler. n.a. = not applicable.

1 2 3 entry compound R = R = R = R = EC50 = IC50 (MTT) =

1 TPC2-A1-P (17) H 2-OCF3, 5-Br CH2cy* CH3 10.5 µM 27 µM

2 SGA-140 (109) CH2CH3 2-OCF3, 5-Br CH2cy* CH3 n.a. 24 µM

3 SGA-43 (96) CH2CH3 - CH2cy* CH3 n.a. 48 µM

4 SGA-50 (100) H - CH2cy* CH3 n.a. 32 µM

5 SGA-48 (110) CH2CH3 2,5-Cl2 CH2cy* CH3 n.a. > 50 µM

6 SGA-52 (116) H 2,5-Cl2 CH2cy* CH3 n.a. 15 µM

7 SGA-54 (97) CH2CH3 2-OCH3, 5-Br CH2cy* CH3 n.a. > 50 µM

8 SGA-55 (115) H 2-OCH3, 5-Br CH2cy* CH3 n.a. > 50 µM

9 SGA-59 (111) CH2CH3 4-F CH2cy* CH3 n.a. 40 µM

10 SGA-66 (117) H 4-F CH2cy* CH3 n.a. > 50 µM

41 RESULTS AND DISCUSSION

11 SGA-61 (112) CH2CH3 4-OCH3 CH2cy* CH3 n.a. 42 µM

12 SGA-67 (118) H 4-OCH3 CH2cy* CH3 n.a. > 50 µM

13 SGA-62 (113) CH2CH3 2,4-F2 CH2cy* CH3 n.a. > 50 µM

14 SGA-68 (119) H 2,4-F2 CH2cy* CH3 n.a. > 50 µM

15 SGA-149 (123) H 2-OCF3, 5-Br pentyl CH3 n.a. 14 µM

16 SGA-150 (125) H 2-OCF3, 5-Br benzyl CH3 34 µM 36 µM

17 SGA-152 (121) H 2-OCF3, 5-Br CH2cy* CH2CH3 n.a. 21 µM

18 SGA-153 (124) H 2-OCF3, 5-Br iPr CH3 n.a. 31 µM

19 SGA-154 (122) H 2-OCF3, 5-Br CH2cy* phenyl n.a. 14 µM

20 SGA-162 (120) H 2-OCF3 CH2cy CH3 n.a. > 50 µM

The toxicity of all substances was examined by in-house MTT assays as described above for TPC2-A1-N (16). Most substances could be classified as non-toxic (SGA-48 (110), SGA-54 (97), SGA-55 (115), SGA-66 (117), SGA-67 (118), SGA-62 (113), SGA-68 (119), SGA-162

(120), entry 5, 7, 8, 10, 12-14, 20, Table 6). Some IC50 values are in a moderate range between

25 and 50 µM (entry 1, 3, 4, 9, 11, 16, 18), and 5 compounds have lower IC50 values (SGA- 140 (109), SGA-52 (116), SGA-149 (123), SGA-152 (121) SGA-154 (122), entry 2, 6, 15, 17,

19, Table 6). TPC2-A1-P (17) itself has an IC50 of 27 µM on HL-60 cells. Recommended working concentration is 30 µM and control experiments are highly relevant to exclude false results.

Only little information was found in the literature about biological activities of TPC2-A1-P-like substances. TPC2-A1-P (17) itself was namely mentioned as a precursor in the synthesis of cannabinoid-1 receptor (CB1R) inverse agonists, whereas the final active compounds contained a carboxamide group instead of the free carboxylic acid function[105].

Phenylpyrrolecarboxamides derived from SGA-50 (100) binding to 5-HT2A, 5-HT2C receptors and the 5-HT transporter were evaluated as antidepressant compounds[103]. This applies only to the carboxamides and there were no data published for the carboxylic acids.

TPC2-A1-P (17) and its 19 analogs were fully analyzed in Fura-2 and Fluo-4 imaging experiments. Only one analog (SGA-150 (125)) was able to activate TPC2 besides the hit 17. No pharmacological targets were published for TPC2-A1-P (17). With its steep structure- activity relationship TPC2-A1-P (17) developed from hit to a promising chemical tool.

42 RESULTS AND DISCUSSION

Comparing the results of both activator classes identified by HTS of the Roche library, a considerable number of TPC2-A1-N analogs caused noteworthy TPC2 activation, but none of them showed significantly increased efficacies or potencies compared to the HTS hit TPC2- A1-N (16). In contrast, the second hit TPC2-A1-P (17) showed an unusually steep structure- activity relationship, and except for one modestly active analog (SGA-150, 125) none of the analogs showed any activity. Thus, the two high-throughput screening hits TPC2-A1-N (16) and TPC2-A1-P (17) can be regarded as strong chemical tools that need further pharmacokinetic and pharmacological characterization.

43 RESULTS AND DISCUSSION

3.4.4 Further results of the novel TPC2 agonists

Within our cooperation with Prof. Dr. Schaefer’s group, Leipzig, we had the opportunity to perform a selectivity screening of the best TPC2 activators identified in our investigations. The selected activators were tested on a broad panel of ion channels of the TRP superfamily. The screening was performed by Nicole Urban in the Schaefer lab (Leipzig).

Figure 16: Selectivity screening of TPC2-A1-N (16), SGA-86 (74), SGA-108 (77), SGA-111 (78) and TPC2-A1-P (17). (A-E) Concentration-effect relationships for Ca2+ increases (Fluo-4 (14)) in response to different concentrations of TPC2-A1-N (16, A), SGA-86 (74, B), SGA-108 (77, C), SGA-111 (78, D) and TPC2-A1-P (17, E). Different stably expressing cell lines were used and activated with the activator indicated. hTPC2, control cells (HEK293), TRPML1 and TRPML3 are highlighted. (F) Legend of the different cell lines used. All experiments were performed by Nicole Urban.

44 RESULTS AND DISCUSSION

At the beginning of the selectivity screening the custom-made fluorescence imaging plate reader was modified and cleaned, which resulted in higher intensities and slightly higher EC50 values (for TPC2-A1-N (16) from 7.8 µM to 11 µM; for TPC2-A1-P (17) from 10.5 µM to 17 µM, Figure 16 A and E). TPC2-A1-P (17), TPC2-A1-N (16) and some of the most potent analogs were analyzed. Earlier experiments already indicated that SGA-85 (73) and SGA-132 (84) had nonspecific activation on control cells (Figure 11 B). The highly substituted analogs SGA-33 (60) and SGA-90 (75) had low solubility which is problematic for experiments using high concentrations. Therefore, these four compounds were excluded. The selected compounds were tested on TPC2, non-transfected HEK-293 cells, the mucolipins TRPML1 and 3, TRPA1, TRPC3-7, TRPM2, 3, 8 and TRPV1-4 (Figure 16 F). Experiments were performed with test concentrations up to 200 µM for analysis of possible side effects.

Comparing the maximum activation, TPC2-A1-P (17) and TPC2-A1-N (16) had the highest efficacy (Figure 16 A and E) and EC50 values within the TPC2-A1-N analogs were all in the same range (9.7 µM to 14 µM, Figure 16 A-D). All tested pre-selected compounds were able to activate TRPA1 at low micromolar concentrations. The activation of TRPA1 was to be expected because TRPA1 is a chemosensory cation channel, which reacts inter alia to chemical substances and generates biological signals[123, 124]. SGA-86 (74) and SGA-111 (78) were able to activate TRPV2 with an EC50 of 46 µM and 19 µM, respectively (Figure 16 B and D). TPC2-A1-P (17) activated TRPV1 at low micromolar concentrations with a very low efficacy (Figure 16 E). The transient receptor potential cation channel subfamily V (“vanilloid”) is related to thermal sensation and responsible for regulating body temperature[125, 126]. SGA-111

(78) was also able to activate TRPM3 with an EC50 of 17 µM (Figure 16 D). Comparing the TPC2-A1-N series, all substances showed effects on other TRP channels in high concentrations (≥ 100 µM). SGA-108 (77) showed the highest selectivity and TPC2-A1-N (16) the highest efficacy. SGA-111 (78) was less specific than all other probes and had a significantly lower efficacy. As mentioned before, all of these compounds have electron- withdrawing groups on the benzoyl moiety and are para-substituted on the anilide side (Figure 12). SGA-108 (77) was the only one with an electron-releasing group on the anilide side, which was generating selectivity for TPC2 at the expense of efficacy. TPC2-A1-P (17) showed high selectivity for TPC2 with high efficacy. The screening further confirmed that potencies were not increased compared to the hits TPC2-A1-N (16) and TPC2-A1-P (17) and despite large efforts in chemical synthesis, these hit structures could not be improved concerning TPC2 activation.

In house agar diffusion test was used to analyze antimicrobial effects of the new compounds on bacteria (Escherichia coli, Pseudomonas marginalis, Staphylococcus equorum, Streptococcus entericus), yeasts (Yarrowia lipolytica, Saccharomyces cerevisiae) and dermatophytes (Hyphopichia burtonii). The compounds did not show any inhibition zones for

45 RESULTS AND DISCUSSION the investigated bacteria and yeasts, while some analogs of TPC2-A1-N and the hit compound itself (16) showed small (but irrelevant) inhibition zones for the dermatophyte Hyphopichia burtonii (Table 7).

Table 7: Inhibition zones for the dermatophyte Hyphopichia burtonii. Agar diffusion experiments were performed by Martina Stadler.

entry compound inhibition zone (Hyphopichia burtonii) 1 TPC2-A1-N (16) 16 mm 2 SGA-1 (44) 12 mm 3 SGA-2 (45) 8 mm 4 SGA-3 (46) 8 mm 5 SGA-4 (47) 8 mm 6 SGA-9 (49) 12 mm 7 SGA-12 (52) 8 mm 8 SGA-16 (55) 10 mm 9 SGA-27 (56) 12 mm 10 SGA-39 (62) 12 mm 11 SGA-70 (64) 12 mm 12 SGA-71 (65) 12 mm 13 SGA-72 (66) 10 mm

3.4.5 Malleable cation selectivity of TPC2

TPC2-A1-N (16) and TPC2-A1-P (17) were further investigated together with different cooperation partners and the results are presented in a recent publication[22]. Both compounds did not activate TPC1 in patch clamp experiments which is stressing the selectivity of these compounds. Cell permeability was proven by GCamp6 experiments, which clearly demonstrated that both activators are able to activate TPC2. The calcium indicator GCamp6, located on the lysosomal membrane, is able to detect calcium efflux from the lysosome, thus proving the membrane permeability of the compounds.

Furthermore, by means of the two novel small-molecules we were able to resolve the conflict

2+ [5, 12, 14, 127, 128] of TPC2 being an NAADP-activated Ca release channel or a PI(3,5)P2 gated Na+ channel[15, 20, 129]. TPC2-A1-N (16) rendered the channel more calcium permeable, similar to NAADP-activated TPC2, whereas TPC2-A1-P (17) increased sodium permeability, similar to the PI(3,5)P2-activated channel. Consequently, ion permeation through TPC2 is ligand- dependent, indicating that TPC2 is a non-selective cation channel with malleable cation

[8] selectivity. Appropriately, the PI(3,5)P2 (4) binding site in TPC2 has been identified to broadly overlapping with the one for TPC2-A1-P (17). It has been demonstrated that TPC2-A1-N (16) induces an alkalinization of single vesicles in cells expressing wild-type TPC2, as previously reported for NAADP (3)[130, 131]. These findings confirmed that TPC2 activation is coupled to

46 RESULTS AND DISCUSSION lysosomal pH and vesicle motility in an agonist-dependent manner. The effect of both activators on lysosomal exocytosis was further evaluated. TPC2-A1-P (17), but not TPC2-A1- N (16) promoted lysosomal exocytosis. Manipulation of lysosomal exocytosis may provide a therapeutic approach for LSDs[132-134].

These findings would not have been achievable without the availability of the lipophilic small- molecule activators TPC2-A1-N (16) and TPC2-A1-P (17) identified in this project. These tools have the potential to elevate studies regarding TPC2 to the next level, which will result in a better understanding of the various ion channel functions.

47 RESULTS AND DISCUSSION

3.4.6 The race to identify new TPC activators

Small-molecule modulators of two pore channels have been a hot topic in recent literature. In 2019, Zhang et al. identified tricyclic and phenothiazine antidepressants as TPC1 and 2 activators by screening Sigma’s LOPAC library, containing 1280 compounds[111]. With calcium imaging experiments, followed by confirmation using whole-cell recordings in TPC2LL/AA- expressing HEK293 cells, the authors identified 8 compounds as TPC2 activators. The dibenzazepine-type tricyclic antidepressants (TCAs) clomipramine (127), desipramine (128), imipramine (129), amitriptyline (130) and nortriptyline (131), as well as the phenothiazine- based antidepressants chlorpromazine (132) and triflupromazine (133) were found to activate

TPC2 (Figure 17 A and B). The EC50 values were between 43 and 112 µM and therefore these activators are presumably less potent than TPC2-A1-N (16, Figure 10 G) and TPC2-A1-P (17, Figure 10 H) from our project. The authors described that currents elicited with TCAs were strongly voltage-dependent while riluzole (134, Figure 17 C) activation was voltage- independent. Some TCAs, clomipramine (127) and desipramine (128), also activated TPC1 in a voltage-dependent manner, while chlorpromazine (132) and riluzole (134) inhibited TPC1. It was barely possible to analyze structure-activity relationships with only seven identified structures. Nevertheless, the dibenzazepine carbamazepine (135) and native phenothiazine (136, Figure 17 D) did not activate TPC2, which highlighted the necessity of the aminoalkyl side chain at the central ring of the tricyclic core[135].

A

N N N Cl

N NH N N NH

clomipramine (127) desipramine (128) imipramine (129) amitriptyline (130) nortriptyline (131)

EC50 = 43 µM EC50 = 87 µM EC50 = 112 µM EC50 = 102 µM EC50 = 52 µM B C D S S S F3CO S N Cl N CF3 NH2 N N N H O NH2 N N riluzole (134) carbamazepine (135) phenothiazine (136) no activation no activation chlorpromazine (132) triflupromazine (133) EC50 = 60 µM EC50 = 63 µM

[111] Figure 17: Structures and EC50 values, published by Zhang et al. . (A-B) Structures and EC50 values of TCAs (A) and related phenothiazines (B) as TPC2 activators. EC50 values were obtained from whole- cell recordings at -140 mV. (C) Structure of riluzole (134). (D) Structures of the inactive carbamazepine (135) and phenothiazine (136).

48 RESULTS AND DISCUSSION

Evaluating the current literature it was noticeable that the phenothiazines triflupromazine (133) and fluphenazine (6), recently published by Penny et al.[31], share the same skeleton but have huge differences in their pharmacological activity (Figure 18). While fluphenazine (6) was

[31] inhibiting TPC2 signals evoked by NAADP (3) with an IC50 of 42 µM , triflupromazine (133) was identified as TPC2 activator. An inhibitor of TPC2 was most likely converted into an activator of TPC2 by introducing only slight changes in the side chain[135]. This example demonstrates how close activators and inhibitors of an ion channel can resemble each other and how important the analysis of structure-activity relationships is.

Figure 18: Structures of the TPC2 activator triflupromazine (133) and the TPC2 inhibitor fluphenazine (6)[31, 111]. Differences are marked in magenta.

The two independently performed HTSs by Zhang et al. and within this project created a panel of small-molecule TPC activators, compared in our recently published review[135]. Zhang et al. focused on drug repurposing and thereby identified TCAs, phenothiazines and the benzothiazole riluzole (134) that activate TPC2. Originally, TCAs are used to treat e.g. depression, bipolar disorder, panic disorder, chronic pain, and insomnia and inhibit inter alia monoamine (serotonin, norepinephrine, dopamine) reuptake. TCAs have a wide range of adverse effects and are therefore replaced as antidepressants by the selective serotonin reuptake inhibitors (SSRI)[136]. In addition, amitriptyline (130), imipramine (129), and clomipramine (127) are also potent CYP450 inhibitors, significantly inhibiting CYP450 2C19 and 1A2[137]. Aforementioned, this bears the risk of undesired drug-drug interactions. Riluzole (134) blocks TTX-sensitive sodium channels, kainite receptors and NMDA receptors, has neuroprotective effects and it is currently approved for the treatment of amyotrophic lateral sclerosis (ALS)[138-142]. The activators identified in this thesis, TPC2-A1-N (16) and TPC2-A1-P (17), are no listed drugs. Comparing the results of both screenings, there is comprehensive knowledge on the pharmacological profiles of the repurposed TCA/phenothiazine-type TPC2 activators due to their long term application in therapy, but as well a large list of undesired effects. Our newly identified activators still need full pharmacokinetic and pharmacological characterization, though have the potential to address TPCs as their main target with a high affinity and thus have less side effects.

49 RESULTS AND DISCUSSION

Lately performed docking studies with the TPC2 agonists on the apo-state hTPC2 structure confirmed that TPC2-A1-P (17) and PI(3,5)P2 (4), as well as TPC2-A1-N (16) and riluzole (134) share one binding pocket, each[135]. This suggests that TPC2-A1-N (16) and riluzole (134) both mimic NAADP (3) actions. Furthermore the binding free energies were calculated and suggest that TPC2-A1-N (16) and TPC2-A1-P (17) are more efficacious than clomipramine (127) and chlorpromazine (132). This corresponds with the higher EC50 values for the repurposed activators (42 – 112 µM) compared to TPC2-A1-N (16, 7.8 µM) and TPC2-A1-P (17, 10.5 µM).

Now there is the opportunity to choose from an impressive and highly diverse collection of new lipophilic small-molecule activators for either selectively activating TPC2 or activation of both TPC1 and TPC2 with the warning that some of the compounds are also blocking TPC1. Physiology and pathophysiology of TPCs can be studied in more detail with the cell permeable small-molecule activators. Most importantly, the novel compounds allow studies in intact cells and can be used as chemical tool for analyzing TPC2 inhibitors. They may also be applicable for in vivo studies and perhaps even for therapy[135].

50 RESULTS AND DISCUSSION

3.5 A new generation of TPC2 inhibitors

A high-throughput analysis of TPC2 inhibitors can be accomplished using the newly developed membrane permeable and small-molecule TPC2 activators. Therefore, a library of TPC2 inhibitors was created to gain first insights into SAR for truncated variants of tetrandrine (1). Aforementioned, our concept was to develop TPC2 inhibitors derived from the lead structure tetrandrine (1). One major intention was to develop less complex analogs of this bisbenzylisoquinoline alkaloid. Hence, we considered both, natural products of this chemotype from different sources as well as monomeric benzylisoquinolines and newly synthesized analogs of tetrandrine (1).

3.5.1 Collection of alkaloids and 1-benzylisoquinolines

Consequently, this library included benzylisoquinoline-type intermediates of the morphine biosynthesis like O,O-dibenzyl coclaurine (Z3, 137), available from the substance collection of Prof. Dr. Meinhart Zenk (passed away in 2011), tetrandrine (1) and derivatives such as fangchinoline (9), kindly provided by Prof. Dr. Peter Pachaly (passed away in 2019), and commercially available benzylisoquinoline alkaloids like dauricine (11) and oxyacanthine (138, Figure 19).

Figure 19: Strutures of investigated bisbenzylisoquinoline alkaloids. (A) Structures of the alkaloids tetrandrine (1), fanglchinoline (9) and N,N-dimethyltetrandrine dichloride (139), kindly provided by Prof. Dr. Peter Pachaly†. (B) Structures of the commercially available alkaloids berbamine dihydrochloride (10), oxyacanthine sulfate (138), cepharanthine (140) and dauricine (11). Structural differences compared to tetrandrine (1) are marked red.

51 RESULTS AND DISCUSSION

The bisbenzylisoquinoline alkaloids, provided from Prof. Dr. Peter Pachaly† were analyzed by NMR, HRMS, specific rotation and purity was confirmed by analytical HPLC. Only tetrandrine (1) from natural source had to be purified by column chromatography and recrystallized from EtOH/water before it was subjected to biological experiments. All analytical data were in accordance with literature[143-145]. All substances from the collection of Prof. Dr. Meinhart Zenk† (Figure 20) were analyzed by NMR, HRMS and specific rotation in order to distinguish (if not clearly indicated) racemic from enantiomerically pure substances. Purity was confirmed by analytical HPLC. If a compound was not pure enough (HPLC purity < 96%), purification was accomplished by FCC. Analytical data for all substances were in accordance to literature or are stated in the experimental part of this thesis.

Figure 20: Structures of substances, kindly received from Prof. Dr. Meinhart Zenk†.

52 RESULTS AND DISCUSSION

3.5.2 Synthesis plan

A new generation of truncated analogs of tetrandrine (1) should be synthesized to receive molecules that resemble tetrandrine (1, Figure 21). A short and efficient synthesis was developed therefore. These molecules should mimic one of the two benzyltetrahydroisoquinoline moieties of tetrandrine (1), therefore the racemic natural product N-methyl coclaurine (SG-132, 159) was chosen as basic structure. Further decoration not only in form of O-benzyl derivatives, but also as diaryl ethers are supposed to closely imitate tetrandrine (1) in shape and size. Bisbenzylisoquinolines with anti-cancer activity have already been described aforementioned. They have different stereochemistry at their two stereocenters, e.g. cepharanthine (S,R, 140)[146] or the seco-variant dauricine (R,R, 11)[80], compared to tetrandrine (S,S, 1), as shown in Figure 19 A and B. Within a first series of endolysosomal patch clamp experiments, performed by Dr. Yu-Kai Chao, antagonistic effects of cepharanthine (140), dauricine (11) and (±)-O,O-dibenzyl coclaurine (Z3 (137), Figure 20) on TPC2 were confirmed (Figure 22). Aforementioned stereochemistry was not limited to one isomer, a racemic synthesis for the truncated analogs of tetrandrine (1) would be most appropriate. Compared to the rigid, macrocyclic bisbenzylisoquinoline tetrandrine (1), the new truncated analogs of tetrandrine bear only one benzylisoquinoline unit. However the O-benzyl or O-phenyl residues mimic the two benzenoid rings of the second benzylisoquinoline moiety of the parent compound making these compounds similar to tetrandrine (1) in size, but are much more flexible. This flexibility should enable the compounds to adapt perfectly to the binding site in the target proteins.

Figure 21: Truncation of tetrandrine (1). The racemic alkaloid (±)-N-methylcoclaurine (159) already represents one half of tetrandrine (1). Introduction of additional aromatic moieties (right, marked in magenta) enables the molecule to resemble the overall molecular geometry of tetrandrine (1).

Key steps for the synthesis of truncated tetrandrine analogs are the N-acyl Pictet-Spengler reaction to construct the 1-benzyltetrahydroisoquinoline and the Chan-Evans-Lam coupling for introduction of diaryl ethers (Figure 21, right). According to Comins et al.[147], the precursors for the N-acyl Pictet-Spengler reaction would be different carbamate building blocks of type I

53 RESULTS AND DISCUSSION and enol ether building blocks of type II. These building blocks would be easily accessible from commercially available aldehydes bearing different residues in meta- and para-position (i, Scheme 16). If desired, the phenol group of these aldehydes can be protected as benzyl ether or alkyl carbonate (ii and v, Scheme 16). The utilization of benzyl ethers as protecting groups is commonly known, while the introduction of alkyl carbonates is new. Following a procedure published by Pouysegu et al.[148], a Henry reaction using commercially available benzaldehydes and nitromethane would give nitrovinyl compounds (iii, Scheme 16) and reduction using lithium alanate would yield desired arylethylamine (iv, Scheme 16). This sequence only needs to be performed for the introduction of benzyl ethers, because many arylethylamines (iv, Scheme 16) are already commercially available (e.g. 3-methoxytyramine

(160) and 3,4-dimethoxyphenyl-ethylamine 161). Using ethyl chloroformate (162) and NEt3 would give the carbamate group and further, in the case of a free phenol group, an ethyl

[149] carbonate (I, Scheme 16) . Depending on the work-up conditions, the ethyl carbonate could remain unaffected using neutral conditions or cleaved using alkaline conditions[73, 149]. The resulting free phenol group of the alkaline work-up could then be used for the introduction of an aryl ether via Chan-Evans-Lam coupling. Wittig olefination of 4-substituted benzaldehydes (i, Scheme 16) would give enol ethers of type II (Scheme 16) as masked arylacetaldehyde.

Scheme 16: Synthesis plan for the preparation of the precursors for the N-acyl Pictet-Spengler reaction. Carbamate building blocks of type I and enol ether building blocks of type II are easily accessible from commercially available aldehydes or arylethylamines.

The 1-benzyl-1,2,3,4-tetrahydroisoquinolines III were to be prepared by N-acyl Pictet- Spengler reactions using precursors I and II (Scheme 17 A). Comins et al. published a procedure for an TFA catalyzed N-acyl Pictet-Spengler reaction, which would be pursued[147]. Utilization of arylethylcarbamates for N-acyl Pictet-Spengler reactions is a common method in

54 RESULTS AND DISCUSSION the Bracher research group. While standard Pictet-Spengler tetrahydroisoquinoline synthesis requires harsh reaction conditions like heating to reflux with strong acids, N-acyl Pictet- Spengler reaction proceeds under mild conditions. This is caused by formation of an N- acyliminium ion 163 as intermediate, which is a much stronger electrophile in comparison to the less powerful electrophile iminium intermediate 164 which occurs in regular Pictet-Spengler reactions (Scheme 17 B). Even nucleophiles that are relatively unreactive as non-activated benzenoids, participate effectively in cyclizations with N-acyliminum species[150, 151].

Scheme 17: The N-acyl Pictet-Spengler reaction. (A) Planed synthesis for the construction of the tetrahydroisoquinoline III via N-acyl Pictet-Spengler reaction. (B) N-Acyliminium ion 163 (left) and iminium ion 164 (right) as intermediates for Pictet-Spengler-type cyclizations.

Tertiary N-methyl amines IV are available via reduction of carbamates III using lithium alanate (Scheme 18)[152]. Furthermore all carbonate esters can be cleaved under these conditions, directly yielding the free phenols and tertiary N-methyl amines in one step, while the benzyl protecting groups of the phenols are not affected by these conditions (not depicted).

Scheme 18: Planned simultaneous deprotection of ethyl carbonates and reduction of carbamates to give N-methyl amines of type IV.

55 RESULTS AND DISCUSSION

For the synthesis of truncated tetrandrine analogs, as depicted in Figure 21, diaryl ethers had to be introduced to the appropriate free phenols. Choosing an environmental-friendly catalyst led to copper-catalyzed coupling reactions. One famous example is the Ullmann coupling for diaryl ether synthesis[153]. Hereby, aryl halides and phenols are converted into diaryl ethers under copper-promotion. Requiring high temperatures and being limited on the substitution patterns of both coupling partners, the Ullmann coupling is not generally applicable, and a new version of this coupling reaction was selected. The Chan-Evans-Lam coupling was developed in 1998 and represents a modern copper-catalyzed oxidative coupling of boronic acids and heteroatom nucleophiles[154-156]. Advantages of the Chan-Evans-Lam coupling are milder reaction conditions (room temperature and the presence of oxygen), cheap copper catalysts, good to excellent yields, and a diverse substrate scope[157].

Scheme 19: Proposed mechanism for the Chan-Evans-Lam diaryl ether synthesis, based on the studies of Stahl and coworkers[158].

A boronic acid dimethyl ester in MeOH was used as example for the studies by Stahl and coworkers of the mechanism of this etherification process[158, 159]. This catalytic cycle starts with a Cu(II) species (a, Scheme 19), in this scheme Cu(OAc)2 and e.g. pyridine as ligand (Ln). Transmetalation with an aryl boronic acid (marked in orange) gives aryl Cu(II) complex (b, Scheme 19). Oxidation of this complex (b, Scheme 19) to a Cu(III) complex (c, Scheme 19) occurs via disproportionation with another equivalent of the starting Cu(II) species (a, Scheme 19). The C-O bond formation takes place within the reductive elimination to give the desired diaryl ether (orange, Scheme 19) and a Cu(I) species (d, Scheme 19), which is re-oxidized by oxygen.

56 RESULTS AND DISCUSSION

Scheme 20: Summary of the synthesis plan for truncated tetrandrine analogs. These analogs (V) can be achieved in three steps, starting from precursors I and II.

Following this sequence would give desired truncated tetrandrine analogs in only three steps, starting from building blocks I and II (Scheme 20). This short sequence was to be used to introduce a variety of different diaryl ethers for the generation of a set of truncated analogs to study SAR.

57 RESULTS AND DISCUSSION

3.5.3 Synthesis of a new generation of TPC2 inhibitors

First, the building blocks for the N-acyl Pictet-Spengler reaction were synthesized. Using ethyl chloroformate (162) and NEt3, amines can be converted into carbamates and phenols into ethyl carbonates. The commercially available 3-methoxytyramine hydrochloride (160) was protected under these conditions and depending on further proceedings yielded carbamate 165 (bearing an ethyl carbonate protecting group) or 166 (having a free phenol group) in high to moderate yields (Scheme 21)[73, 149]. While neutral extraction gave the protected phenol 165, the addition of NaOH (1 M) in ethanol deprotected the aryl-alkyl carbonate and resulted in phenol 166. If the reaction mixture was extracted using 2 M aq. NaOH, the deprotection of carbamate 165 was not complete, because of the two-phase mixture.

Scheme 21: Synthesis of carbamates 165 and 166 using ethyl chloroformate (162) under basic conditions. Depending on the work up, a protected phenol 165 or deprotected phenol 166 was obtained.

Diaryl ether 167 was synthesized using the mild coupling conditions of the Chan-Evans-Lam reaction. Copper catalyzed and under alkaline conditions carbamate 166 and phenylboronic acid (168) were used to give diary ether 167 in high yields (Scheme 22). Full characterization (TLC, NMR, HRMS, IR, m.p.) confirmed the structure and analytical HPLC the purity. Initial experiments for this reaction were performed within the Bachelor thesis of F. Talay.

Scheme 22: Copper catalyzed Chan-Evans-Lam coupling of carbamate 166 to give diaryl ether 167.

58 RESULTS AND DISCUSSION

The aforementioned method to generate carbamates was applied for the synthesis of carbamate 169 as well, with commercially available 3,4-dimethoxyphenylethylamine 161 as starting material. The reaction time was reduced to 4 h by refluxing the mixture to give carbamate 169 in high yields (Scheme 23). Carbamate 169 was fully characterized (TLC, NMR, HRMS, IR, m.p.) and 1H and 13C NMR data were in accordance with literature[160].

Scheme 23: Protection of amine 161 with accelerated reaction time.

To receive an O-benzylated carbamate (170, Scheme 25), the corresponding amine 171 had to be synthesized first. Following a procedure published by Pouysegu et al.[148], 4-benzyloxy- 3-methoxybenzaldehyde 172 was used as starting material for a Henry reaction, giving nitrovinyl compound 173 in good yield. Reduction using lithium alanate yielded the desired arylethylamine 171 in high yield (Scheme 24). Both products were fully characterized and analytical data are in accordance with literature.

Scheme 24: Synthesis of benzyl-protected amine 171 in two steps, according to Pouysegu and colleagues[148].

Using ethyl chloroformate (162) and NEt3, amine 171 was protected, giving carbamate 170 in moderate yield (Scheme 25). The compound was characterized by TLC, NMR, HRMS, IR and melting point and the analytical data were in accordance with literature[149].

Scheme 25: Synthesis of carbamate 170 utilizing the above mentioned method.

59 RESULTS AND DISCUSSION

Enol ethers were utilized as masked arylacetaldehyde equivalents, because the corresponding arylacetaldehydes are often instable due to their ability to easily polymerize[161]. The E/Z stereochemistry of the enol ethers is not relevant for the following N-acyl Pictet-Spengler reaction and a simple Wittig olefination of aromatic aldehydes was chosen for the synthesis. Enol ether 174 was to be formed starting with 4-hydroxybenzaldehyde (175), (methoxymethyl)- triphenyl-phosphonium chloride and a strong base (Scheme 26). First, the phosphonium salt is deprotonated to give a colorful ylide, which then forms an oxaphosphetane via a [2+2] cycloaddition with the aldehyde. Elimination of triphenylphosphine oxide yields the desired E/Z- alkene. The synthesis of enol ether 174 is known using tert-butoxide (conditions a)[162] or LHMDS (conditions b)[163]. Though, the first step, the ylide formation, did not show any conversion for potassium tert-butoxide and only slight conversion using the stronger base LHMDS. The even stronger base LDA (conditions c) was further tried and finally ylide formation was observed by formation of a deep red complex. Both known procedures described high yields for the Wittig olefination (81% and >95%), however, this was not reproducible. Formation of the enol ether 174 was observed via GC-MS but not completed even after 24 h, therefore another route was chosen.

Scheme 26: Synthetic approach to yield enol ether 174, using different conditions.

An ethyl carbonate protecting group for phenols was already used for some carbamate building blocks and was now introduced for protection of the free phenolic group of aldehyde 175.

Hence 4-hydroxybenzaldehyde (175) was protected using ethyl chloroformate (162) and NEt3 to give aldehyde 176 in high yield (Scheme 27).

Scheme 27: Synthesis of protected aldehyde 176 using ethyl chloroformate (162) and NEt3.

Aforementioned formation of the ylide for Wittig olefination was most successful using LDA. Hence LDA was used for olefination reaction of aldehyde 176. With (methoxymethyl)-

60 RESULTS AND DISCUSSION triphenylphosphonium chloride and aldehyde 176, enol ether 177 was obtained in good yield (Scheme 28). The reaction was monitored by TLC and staining with DNPH as both starting material and product had the same Rf value. DNPH indicated the end of the reaction by different colors of the formed hydrazones. While the aromatic, highly conjugated aldehyde 176 resulted in a deep orange color, the aliphatic enol ether 177 gave a lighter, yellow color. The generation of a side product, enol ether 174, was observed and starting material 175 was found as well. The ethyl carbonate protecting group is labile under alkaline conditions and therefore only one equivalent LDA was used. Maybe the basicity of the formed ylide is strong enough to cleave the ethyl carbonate protecting group and yield enol ether 174 or aldehyde 175 (if the starting material was deprotected). Consequentially, the reaction cannot result in a full conversion of aldehyde 176 into enol ether 177, as not enough ylide was present. Both side products were separated from the desired enol ether 177 by column chromatography. While being stored the enol ether 177 decomposes, as already described for enol ether 174[162]. Therefore it was analyzed by TLC, NMR and HRMS as fast as possible and quickly used for the next step without full characterization.

Scheme 28: Wittig olefination of aldehyde 176. Besides the desired enol ether 177, also two side products were identified.

This method was further applied for other para-substituted benzaldehydes. Besides para- carbonate protecting group in enol ether 177 (entry 1, Table 8), a diaryl ether enol ether 178 (entry 2, Table 8) and a benzyloxy enol ether 179 (entry 3, Table 8) were synthesized in moderate to high yields. Due to the instability of enol ether 177 and the reports in literature about it[162], all of them were analyzed by TLC, NMR and HRMS as fast as possible and used without full characterization for the next step.

61 RESULTS AND DISCUSSION

Table 8: Wittig olefination of different aldehydes to give enol ethers using (methoxymethyl)-triphenyl- phosphonium chloride and LDA as base.

entry starting material reaction time product yield

1 R = COOEt (176) 16 h 63%

2 R = Ph 50 h 59%

3 R = Bn 16 h 85%

With the four different carbamates of type I and the three enol ethers of type II (Scheme 20) both building blocks for N-acyl Pictet-Spengler reactions were successfully synthesized and ready to be combined.

For performing the N-acyl Pictet-Spengler reaction TFA in dichloromethane was chosen as acidic catalyst, according to Comins et al.[147]. Molecular sieves (4 Å) were added to ensure an aqueous free environment and avoid side or decomposition reactions. These conditions gave racemic tetrahydroisoquinoline 180 in high yield (Scheme 29). NMR analysis was performed at 100 °C in tetrachloroethane. In NMR experiments performed at room temperature, different rotamers of the molecule were observed. This is the result of isomers arising from hindered single-bond rotation in a time frame that it is detectable by NMR. Heating up the NMR sample to 100 °C boosted the rotation to give a single set of resonances.

Scheme 29: N-Acyl Pictet-Spengler reaction to give racemic tetrahydroisoquinoline 180. Carbamate 165 and enol ether 177 were used as starting materials.

These reaction conditions were further applied in the synthesis for racemic tetrahydroisoquinolines (III, Table 9) generated out of different combinations of the synthesized carbamates and enol ethers (I and II, Table 9). As precursor for the natural

62 RESULTS AND DISCUSSION product (±)-N-methylcoclaurine (159) served the ethyl carbonate protecting group (entry 1, Table 9). To analyze the influence of residues in C-7 position of the tetrahydroisoquinoline a methoxy group (entry 2 and 5, Table 9), a diaryl ether (entry 3, Table 9) and a benzyl ether (entry 4, Table 9) were introduced. The O-benzylated tetrahydroisoquinoline (entry 4, Table 9) was more complex to purify because FCC mainly gave impure fractions. Hence this yield is lower. All other N-acyl Pictet-Spengler reactions gave the racemic tetrahydroisoquinolines in high yield. NMR analysis at 100 °C (to avoid rotameric NMR resonances), TLC, HRMS and IR confirmed the identity and analytical HPLC the purity.

Table 9: Synthesis of various racemic 1-substituted tetrahydroisoquinolines (III) starting from different carbamate and enol ether building blocks (I and II) using N-acyl Pictet-Spengler reactions. *Initial experiments for this reaction were performed within the Bachelor thesis of F. Talay.

reaction entry carbamate I enol ether II product III yield time

1 18 h 81%

2 20 h 77%

3 90 h 90%*

4 18 h 32%

5 20 h 92%

63 RESULTS AND DISCUSSION

[152] Reduction of the carbamate group of tetrahydroisoquinolines III (Table 10) using LiAlH4 directly yielded the corresponding N-methyl compounds. Preparation of the corresponding secondary amines from the carbamate intermediates is feasible in general, but would need other reaction conditions, like carbamate hydrolysis using KOH and hydrazine hydrate at 120 °C for many days[164] or refluxing in aq. 10 M HCl solution overnight[165]. Though, tertiary amines were preferred, as they showed more resemblance to the structure of tetrandrine (1). Furthermore ethyl carbonate protecting groups were cleaved one-pot within the reduction of the carbamate using lithium alanate, resulting in a number of N-methyl compounds (IV, Table 10).

A procedure published by Cava et al. was applied for the synthesis of N-methyl amines of type

IV (Table 10) by refluxing carbamates (III, Table 10) with an excess of LiAlH4 under dry conditions in THF. For reaction work-up avoiding undesirable treatment with water under formation of aluminum hydroxide gels Glauber’s salt was chosen, which is Na2SO4 x 10 H2O.

The crystal water of this salt causes a slow and moderate decomposition of the LiAlH4 excess and prevents uncontrolled heating of the mixture. The reaction was finished when no gas evolution could be observed anymore. After filtration the product was purified by extraction using the phase swap technique in which the crude organic product phase was extracted with 2 M aq. HCl solution to transfer the protonated amine into the aqueous phase. Then, neutralization of the aqueous phase yielded the tertiary amine again and re-extraction with organic solvents resulted in the pure product. This technique avoids the more time-consuming FCC, especially for upscale reactions. By this means all synthesized carbamates (III) were reduced to the corresponding tertiary N-methyl amines (IV, Table 10) in moderate to high yields. Two natural products ((±)-N-methylcoclaurine (159) and (±)-armepavine (185)) were synthesized in their racemic form this way (entry 1, 2, Table 10), one derivative of a natural product (entry 4, Table 10) and two new synthetic tetrahydroisoquinolines (entry 3, 5, Table 10). Full characterization (TLC, NMR, HRMS, IR, m.p.) confirmed the structure and analytical HPLC the purity of the products.

64 RESULTS AND DISCUSSION

Table 10: Reduction of the carbamate function and deprotection of carbonic acids of tetrahydroisoquinolines (II) using LiAlH4 gave N-methyl amines of type IV. * Initial experiments for this reaction were performed within the Bachelor thesis of F. Talay.

starting material reaction name of natural entry product IV yield III time product or derivative

R = COOEt 1 R’ = COOEt 3 h (±)-N-methylcoclaurine 79% 180

R = Me 2 R’ = COOEt 3 h (±)-armepavine 35% SG-089 (181)

R = Ph 3 R’ = COOEt 5 h - 79%* 182

R = Bn (±)-O,O-dibenzyl N- 4 R’ = Bn 18 h 46% methylcoclaurine SG-145 (183)

R = Me 5 R’ = Ph 20 h - 46% 184

Synthesis of (±)-armepavine (185) had the lowest yield (entry 2, Table 10). A reason for this is, that a second tetrahydroisoquinoline (189) was isolated (Scheme 30 A and B). The N-acyl Pictet-Spengler reaction as well as the lithium alanate reduction were both performed in large scale (22.1 mmol and 16.9 mmol) and to improve time efficacy purification was reduced to a minimum. Therefore the enol ether 177 also had to be synthesized in large scale and aforementioned side products (aldehyde 175 and enol ether 174, Scheme 30 B) occurred. These two side products were both able to act as starting material for the N-acyl Pictet- Spengler reaction to give 1-benzyl-carbamate 190 and 1-phenyl-carbamate 191 (Scheme 30 B). Reduction of 1-benzyl-carbamate 190 would also result in SG-121 (185) and was therefore not removed. Thus, 1-phenyl-carbamate 191 was carried over as an unrevealed impurity and

65 RESULTS AND DISCUSSION following reduction of a mixture of three different carbamates (181, 190 and 191) yielded SG- 121 (185) and side product SG-121-NP (189). The two products were separated by FCC, analyzed and purity was confirmed by analytical HPLC.

Scheme 30: Synthesis of tetrahydroisoquinolines 185 and 189. (A) Synthesis of (±)-armepavine (185), as described above. (B) Side products of the Wittig olefination 174 and 175 underwent N-acyl Pictet- Spengler reaction to yield carbamates 190 and 191. Following lithium alanate reduction yielded (±)- armepavine (SG-121, 185) and side product SG-121-NP (189). Shaded products were not isolated within this sequence.

Utilizing the method developed by Chan, Evans and Lam many diaryl ethers (V, Table 11) were synthesized. Per phenolic group of the amine (IV, Table 11) 1 equivalent copper acetate,

3 equivalents arylboronic acid (VI, Table 11) and a mixture of NEt3 and pyridine (1:1, 2.5 eq. each) were used. The resulting oily tertiary amines were characterized and then treated with methanolic HCl solution to give the amorphous hydrochloride salts as solids which were used for biological experiments. Two diaryl ethers were recrystallized as hydrochloride salts (entry 3, 9, Table 11) and were also characterized such. The diaryl ether variant of N- methylcoclaurine, SG-094 (192), was the first to be synthesized using the double amount of equivalents for the two phenolic groups of SG-132 (159, entry 1, Table 11). Two diaryl ethers with a methoxygroup in C-7 position of the tetrahydroisoquinoline (193 and 194, entry 2, 3, Table 11) and the 1-phenyl-tetrahydroisoquinoline 195 (entry 4, Table 11) were synthesized to study the loss of one phenyl group. Tetrandrine (1) has four methoxy moieties, which inspired the synthesis of compounds bearing methoxy residues (196 and 197, entry 7, 8, Table 11) or replaced them by the metabolically stable bioisosteric trifluoromethoxy group (198, entry 6, Table 11). Electron releasing groups were also introduced (199 and 200, entry 5, 9, Table 11). A nitrile residue was also part of the structure of the TPC2 activator TPC2-A1-N (16). Hence a nitrile residue was introduced to probably have a better affinity to the ion channel (201, entry 11, Table 11). A 3,4-dichloro substituted aromatic ring was synthesized to study

66 RESULTS AND DISCUSSION the influence of halides (202, entry 12, Table 11). To analyze the influence of heterocyclic aromatic residues, a pyridine moiety was introduced (203, entry 10, Table 11), whereas the synthesis of a variant bearing a furan moiety (204) was not successful (entry 14, Table 11), which is in accordance with one report in literature[166]. Same applied to the tetrahydroisoquinoline 205, which should have been synthesized to be labeled by click chemistry (entry 13, Table 11). Overall 12 tetrahydroisoquinolines bearing a diaryl ether motif were synthesized in moderate to high yield and fully characterized by TLC, NMR, HRMS and IR (melting points only for the hydrochloric acid salts) and purity was affirmed by analytical HPLC.

Table 11: Products of the Chan-Evans-Lam reaction. Using different arylboronic acids (VI) and a variety of racemic amines of type IV yielded the desired diaryl ethers of type V.

starting material boronic acid reaction product entry yield IV VI time V

R,R’ = H 1 R’’ = H 18 h 70% SG-132 (159)

R = Me

2 R’ = H R’’ = 4-OCH3 18 h 88% SG-121 (185)

R = Me R’’ = 3,4,5- 3 R’ = H 18 h 84% (OCH3)3 SG-121 (185)

R = Me

4 R’ = H R’’ = 4-OCH3 18 h 95% SG-121-NP (189)

67 RESULTS AND DISCUSSION

R = Ph

5 R’ = H R’’ = 4-CF3 18 h 24% 186

R = Ph

6 R’ = H R’’ = 4-OCF3 13 h 47% 186

R = Ph

7 R’ = H R’’ = 4-OCH3 20 h 67% 186

R = Ph R’’ = 3,4,5- 8 R’ = H 20 h 69% (OCH3)3 186

R = Ph

9 R’ = H R’’ = 4-CH3 20 h 48% 186

R = Ph pyridine-4-boronic 10 R’ = H 20 h 25% acid 186

R = Ph 11 R’ = H R’’ = 4-CN 20 h 48% 186

R = Ph

12 R’ = H R’’ = 3,4-Cl2 20 h 78% 186

R = Ph 13 R’ = H R’’ = 4-C≡C 20 h - 186

R = Ph furan-2-ylboronic 14 R’ = H 20 h - acid 186

68 RESULTS AND DISCUSSION

The compound collection provided by Prof. Dr. Meinhart Zenk† included racemic O,O- dibenzylated coclaurine (Z3, 137, Scheme 31 A) and racemic O,O-dibenzylated nororientaline (Z6, 145, Scheme 31 B). Utilizing these secondary amines, N-methyl amines were obtained by Eschweiler-Clarke reaction[167]. Iminium formation with formaldehyde, followed by reduction with a hydride source yielded tertiary amines. Originally formic acid acted as hydride source, while modified versions used sodium cyanoborohydride. Using the latter yielded SG-005 (187) and SG-159 (206) in moderate yields, although long reaction times did not lead to complete consumption of the starting material (Scheme 31 A and B).

Scheme 31: Eschweiler-Clarke reaction for the synthesis of the N-methyl compounds SG-005 (187, A) and SG-159 (206, B).

Another method for the synthesis of SG-005 (187) was the Mitsunobu reaction with SG-132 (159) as starting material (Scheme 32). Mitsunobu conditions prevented the formation of a quaternary ammonium salt 207, which was the result of using standard protecting conditions (excess of benzyl chloride and a base) for the phenolic groups (Scheme 32). Monitoring via TLC indicated the formation of SG-005 (187), though after 18 h only the quaternary ammonium salt 207 was found. Mitsunobu reaction, however, gave SG-005 (187) in moderate yield and analytical data were in accordance with the analytical data of the two other routes.

69 RESULTS AND DISCUSSION

Scheme 32: Mitsunobu reaction of SG-132 (159) with benzyl alcohol yielded SG-005 (187). Protection of SG-132 (159) using benzyl chloride gave quaternary amine 207.

Synthesis of an N-ethyl variant of bis(benzyloxy)-orientaline 208 was performed using bromoethane in alkaline conditions. Potassium iodide was added for an in situ Finkelstein reaction to boost the conversion rate. TLC monitoring did not show consumption of the starting material, because product and starting material had the same Rf value. ASAP showed the masses of starting material and product and after 6 days no changes of the conversion rate were observed any more. Purification needed semi-preparative HPLC, because standard FCC did not separate secondary amine 145 from N-ethyl amine 208. This resulted in pure SG-158 (208, Scheme 33) in moderate yield. A Leuckart-Wallach reaction[168] could be tried to improve the conversion rate. This reaction is related to the Eschweiler-Clarke reaction but is not limited to formaldehyde. Though, the first reaction yielded enough SG-158 (208) and repetition was not necessary.

Scheme 33: Synthesis of SG-158 (208) with Z6 (145) as starting material.

A total amount of 16 N-alkyl amines was synthesized, including 3 O-benzylated compounds and 13 diaryl ethers. Analysis (TLC, NMR, HRMS, IR and melting points) confirmed the structure and analytical HPLC affirmed the purity of these molecules. All of them resemble one

70 RESULTS AND DISCUSSION half of tetrandrine (1) plus the two benzenoid rings of the second benzylisoquinoline moiety and were further investigated on their biological activity. The synthetic route furthermore generated intermediates that therefore served as negative controls, as SG-132 (159) and SG- 145 (183).

3.6 Pharmacological investigation of TPC2 antagonists

The effect of the inhibitors of TPC2 was again analyzed by Fura-2 and Fluo-4 based calcium imaging as well as by endo-lysosomal patch clamp experiments. Furthermore, their ability to combat diseases like cancer or virus infections1 was investigated. Main focus was on tetrandrine (1), SG-005 (187) and SG-094 (192), because these emerged as the most interesting substances. All studies were performed in close cooperation with Prof. Dr. Michael Schaefer’s group (Fluo-4 based experiments, performed by Nicole Urban), Prof. Dr. Angelika Vollmar’s group (cancer investigation, performed by Martin Müller) and Prof. Dr. Dr. Christian Grimm’s as well as Prof. Dr. Martin Biel’s groups (Fura-2 based calcium imaging, performed by myself and patch clamp experiments, performed by Dr. Yu-Kai Chao).

3.6.1 First identification of the truncated TPC2 inhibitors

The whole endo-lysosomal patch clamp technique is state-of-the-art to confirm direct effects on the activity of endo-lysosomal ion channels[169]. In brief, endolysosomes are enlarged using vacuolin-1, the membrane is destroyed with a glass pipette and then the enlarged endo- lysosomal organelle is carefully isolated. Having the isolated organelle allows the operator to apply hydrophilic, not membrane permeable compounds like PI(3,5)P2 (4) directly to the endolysosome to activate the ion channel. The addition of a potential inhibitor after activation then directly shows the effect of the compound. This unique technique is very accurate but highly complex and requires much time and effort.

Tetrandrine (1) has been known to inhibit TPC2 since 2015[2] and was therefore analyzed as a reference. Thus, by applying the endo-lysosomal patch clamp technique, several other bisbenzylisoquinoline alkaloids were also identified as TPC2 inhibitors. Fangchinoline (9), cepharanthine (140) and the seco-analog dauricine (11) were virtually equipotent to tetrandrine (1, 54% inhibition, Figure 22 A). Fangchinoline (9) is a nor-derivative of tetrandrine (1) and is already known to be a TPC2 inhibitor (Figure 22 D)[30]. Cepharanthine (140) differs from tetrandrine (1) in the absolute stereochemistry and the connection of both benzyl residues and dauricine (11) is no macrocyclic compound (Figure 22 D). This gave a first hint that

1 Studies for virus infections are still in the early stages and not further discussed.

71 RESULTS AND DISCUSSION stereochemistry, the substitutions pattern of lead structure tetrandrine (1) and the fact that tetrandrine (1) is a macrocyclic ring is not mandatory for TPC2 inhibition. Racemic coclaurine (Z1, 141) had nearly no effect, whereas racemic O,O-bisbenzylcoclaurine (Z3, 137) was equipotent to tetrandrine (1, Figure 22 A and E). Contrary, Z6 (145) showed an (on top)- activating effect (Figure 22 A and E). Notably, the bis-phenyl ether SG-094 (192) significantly increased the percentage of channel inhibition (75%) compared to tetrandrine (1, 54%), whereas the corresponding bis-benzyl ether SG-005 (187) showed nearly the same inhibition (44%) as tetrandrine (1, Figure 22 B and Figure 23 H). Both compounds are derived from the racemic alkaloid coclaurine (159).

Figure 22: Patch clamp results to identify TPC2 inhibitors, performed by Dr. Yu-Kai Chao. (A-B) The inhibition percentage of several TPC2 blockers is displayed. Inhibitors (10 µM) were applied upon activation with PI(3,5)P2 (4, 1 µM) on isolated and vacuolin-enlarged endolysosomes from HEK293 cells expressing TPC2-EGFP. The bar graph indicates mean ± SEM of n independent experiments. *p < 0.05, ns = not significant, using unpaired students t-test. (C) Representative current density – voltage relation for a recording of the most potent TPC2 inhibitor of this set of compounds, SG-094 (192), is shown. (D) Structures of tetrandrine (1), fangchinoline (9), cepharanthine (140) and dauricine (11). Differences to tetrandrine (1) are marked in red. (E) Structures of Z1 (141), Z3 (137) and Z6 (145).

The two lipophilic, membrane-permeable TPC2 activators TPC2-A1-N (16) and TPC2-A1-P (17), developed in the first part of this project, allowed to also test inhibitory activities of the compounds in calcium imaging experiments. Fura-2 based single cell calcium imaging experiments were performed first. The best option to assess inhibitory effects is to first add the inhibitor to the cells and incubate for a defined time, then stimulate the cells with the activator and monitor effects over time in the constant presence of the inhibitor. As control, a subsequent experiment needs to be performed in which vehicle, e.g. DMSO only is added first, followed by

72 RESULTS AND DISCUSSION addition of the agonist. The results of these two experiments are then directly compared. If activation levels do not show differences, the substance has no inhibitory activity. At least three independent experiments should be performed for quantification and statistical analysis. Of note, results strongly depend on the transfection levels and these may vary. For more consistent experiments HEK-293 cells stably expressing the plasma membrane variant of hTPC2 were therefore used.

This method was applied for tetrandrine (1), two of the synthesized truncated variants, SG-005 (187) and SG-094 (192), and the controls SG-132 (159) and SG-145 (183). As expected, tetrandrine (1), SG-005 (187) and SG-094 (192) were able to inhibit TPC2-A1-N (16) induced activation significantly (Figure 23 A-D). SG-132 (159) is lacking the prolonging aromatic groups (benzyl or diaryl ethers) while SG-145 (183) has two benzyl ethers but the tetrahydroisoquinoline nitrogen is not basic. Both substances were not able to inhibit TPC2- A1-N (16) induced activation (Figure 23 E-G) and confirmed that the absent groups were essential for the inhibitory effect on TPC2.

Figure 23: Reversed single cell calcium imaging experiments to identify TPC2 inhibitors. (A) Fura-2 based Ca2+ imaging results showing the effect of TPC2-A1-N (16, 10 µM) after stimulation with tetrandrine (1, 10 µM), SG-094 (192, 10 µM), SG-005 (187, 10 µM) or a DMSO control. HEK293 cells stably expressing hTPC2L11A/L12A-RFP were used. Mean values normalized to basal (400 s after activation) ± SEM of at least three independent experiments with 3–10 cells each are shown. (B-D) Representative Ca2+ signals for experiments as in (A). Cells were sequentially stimulated with tetrandrine (1, 10 µM, B), SG-094 (192, 10 µM, C), SG-005 (187, 10 µM, D) or DMSO (0.5% DMSO in HBS) and the activator TPC2-A1-N (16, 10 µM, 400 s). (E) Experiments as in (A) using SG-132 (159, 10 µM), SG-145 (183, 10 µM) or a DMSO control. (F-G) Representative Ca2+ signals for experiments as in (E). Cells were sequentially stimulated with SG-132 (159, 10 µM, C), SG-145 (183, 10 µM, D) or DMSO (0.5% DMSO in HBS) and the activator TPC2-A1-N (16, 10 µM, 400 s). **p < 0.01, *p < 0.05, ns = not significant, using one-way ANOVA followed by Tukey’s post hoc test. In all experiments highlighted

73 RESULTS AND DISCUSSION lines represent the mean response from a population of cells. Shaded traces represent responses of single cells. All experiments were performed on a Leica DMi8 live cell microscope. (H) Structures of SG- 005 (187) and SG-094 (192). (I) Structures of SG-132 (159) and SG-145 (183).

This set of experiments proved the accuracy of the method and the previously obtained electrophysiology results. Of the three inhibitors again SG-094 (192) had the strongest effect (**) compared to tetrandrine (1, *) and SG-005 (187, *) when applied at a concentration of 10 µM (Figure 23 A-D). In a next step the potencies of the compounds were determined by concentration-effect relationship measurements using the HTS system instead of using only single concentrations.

3.6.2 Analysis of the compound library

All compounds were analyzed by using the Fluo-4 based calcium imaging method (HTS system) described before. The experiments were again performed in the group of Prof. Dr. Michael Schaefer (Leipzig) by sequential application of inhibitors at different concentrations followed by the agonists TPC2-A1-P (17) and TPC2-A1-N (16).

Figure 24: Fluo-4 calcium imaging experiments of the highlighted compounds tetrandrine (1), SG-005 (187) and SG-094 (192). Concentration-effect relationships using HEK293 cells stably expressing hTPC2L11A/L12A-RFP are presented. Cells were sequentially stimulated with inhibitor (100 µM → 0.1 µM) and TPC2-A1-N (16, 10 µM) or TPC2-A1-P (17, 10 µM) as activator. IC50 values were calculated out of at least three independent experiments using GraphPad. All experiments were performed by Nicole Urban.

Both activators were used at a concentration of 10 µM. All three inhibitors were found to have

IC50 values in the range of 2.5 µM – 24 µM (Figure 24 A). Significant differences in potencies were not detectable. Surprisingly, SG-005 (187) showed a reduced maximum efficacy (only

74 RESULTS AND DISCUSSION

50%) after activation with TPC2-A1-N (16), while tetrandrine (1) and SG-094 (192) approximated a complete inhibition (nearly 100%, Figure 24 A). All three inhibitors had nearly 100% blocking efficacy after activation with TPC2-A1-P (17).

Figure 25: Structures of the truncated tetrandrine analogs, analyzed in SAR. (A) Structures of the 1- benzyl-tetrahydroisoquinolines synthesized within this project. (B) Structures of the 1-benzyl-tetrahydro- isoquinolines, provided by Prof. Dr. Meinhart Zenk†.

IC50 values were obtained for the other compounds as well (Table 12). For some Z compounds, unspecific effects were observed at concentrations above 50 µM and concentration-effect relationships were not applicable. Tetrandrine (1), fangchinoline (9), berbamine (10), cepharanthine (140), oxyacanthine (138) and dauricine (11) all showed IC50 values in the same range for both activators (entry 1, 4-8, Table 12, all depicted in Figure 19). These results

75 RESULTS AND DISCUSSION confirmed that the substitution pattern of the benzyl residue and the stereochemistry were not determined. SG-005 (187), SG-094 (192), SG-158 (208) and SG-159 (206) showed IC50 value in the same range for activation with TPC2-A1-P (17) while only SG-094 (192) had a slightly higher IC50 for activation with TPC2-A1-N (16, entry 2-3, 17-18, Table 12, structures in Figure

25). Both compounds selected as controls did either show high IC50 values or fitting was not applicable at all (entry 12-13, Table 12, structures in Figure 25). All analogs of SG-094 (192) blocked TPC2 activation with both activators to some extent, while some had differences in potencies (entry 9-11, 14-24, Table 12, structures in Figure 25). The substances bearing a para-methoxy, para-methyl or their fluorinated variants (SG-153 (199), SG-154 (198), SG-155

(196), SG-162 (200)) showed a lower IC50 value after activation with TPC2-A1-P (17) compared to activation with TPC2-A1-N (16) (entry 14-16, 21, Table 12, structures in Figure 25). None of the further analogs showed significantly stronger effects on TPC2. The three Z substances that did not show unspecific effects, Z1 (141), Z9 (147) and Z22 (157) were not able to inhibit TPC2 as well, which was verified by high (>50 µM) or not applicable IC50 values (entry 25-27, Table 12, structures in Figure 25). All other compounds, mainly of the Z series, had a slight inhibitory effect on the channel (at 12.5 µM). These compounds were not of further interest because of their already mentioned nonspecific effects on stably transfected HEK293 cells.

Table 12: Screening results of the compound library using Fluo-4 based calcium imaging and toxicity screening using MTT assay. Concentration-effect relationships were generated using HEK293 cells stably expressing hTPC2L11A/L12A-RFP and were plotted using GraphPad. Experiments were performed as described in Figure 24 in duplicates of one replicate for each activator. Calcium imaging experiments were performed by Nicole Urban and MTT assay by Martina Stadler. **was activated with 30 µM TPC2- A1-P (17).

IC50 (Fluo-4; IC50 (Fluo-4; entry compound IC50 (MTT) TPC2-A1-P (17)) TPC2-A1-N (16)) 1 tetrandrine (1) 10 µM 14 µM 43 µM 2 SG-005 (187) 7.6 µM 2.5 µM 11 µM 3 SG-094 (192) 8.3 µM 24 µM 23 µM 4 fangchinoline (9) 11 µM 7.0 µM 38 µM 5 berbamine HCl (10) 10 µM 7.0 µM 21 µM 6 cepharanthine (140) 8.1 µM 5.1 µM 25 µM 7 oxyacanthine sulfate (138) 21 µM 4.5 µM >50 µM 8 dauricine (11) 12 µM 8.6 µM >50 µM 9 SG-083 (188) 11 µM 14 µM 8.2 µM 10 SG-122 (193) 14 µM 7.3 µM 22 µM 11 SG-127 (194) n.a. 25 µM 18 µM 12 SG-132 (159) >50 µM** 45 µM >50 µM 13 SG-145 (183) 43 µM n.a. >50 µM 14 SG-153 (199) 22 µM >50 µM 15 µM 15 SG-154 (198) 12 µM >50 µM 9.5 µM 16 SG-155 (196) 15 µM 37 µM 14 µM 17 SG-157 (195) 34 µM 30 µM 29 µM 18 SG-158 (208) 9.6 µM 0.33 µM 12 µM

76 RESULTS AND DISCUSSION

19 SG-159 (206) 9.9 µM 0.75 µM 14 µM 20 SG-161 (197) 10 µM 24 µM 15 µM 21 SG-162 (200) 19 µM >50 µM 16 µM 22 SG-163 (203) 18 µM 21 µM 9.5 µM 23 SG-164 (201) 15 µM 43 µM 21 µM 24 SG-165 (202) 33 µM >50 µM 19 µM 25 Z1 (141) >50 µM >50 µM >50 µM 26 Z9 (147) >50 µM >50 µM >50 µM 27 Z22 (157) n.a. >50 µM 29 µM

For acute toxicity, MTT assays were again performed by Martina Stadler. For these experiments HL-60 cells were used, which are human leukemia cells. All compounds showed acute toxicity to HL-60 cells. That was to be expected because of the strong correlation between cancer and TPC2[36]. The compounds that were not able to block TPC2 also showed no (entry 12-13, 25-26, Table 12) or slight (entry 27, Table 12) toxicity on HL-60 cells. Interestingly, the TPC2 inhibitors oxyacanthine (138) and dauricine (11) were not cytotoxic as well (entry 7-8, Table 12).

In house agar diffusion test (Martina Stadler) was used to exclude some side-effects on bacteria (Escherichia coli, Pseudomonas marginalis, Staphylococcus equorum, Streptococcus entericus), yeasts (Yarrowia lipolytica, Saccharomyces cerevisiae) and dermatophytes (Hyphopichia burtonii). All compounds of the SG-series (Figure 25) did not show an inhibition zone.

A variety of TPC2 inhibitors were identified within this screening. SG-005 (187) and SG-094

(192) emerged to be the most promising TPC2 inhibitors with low IC50 values and high efficacies. Decorations on the aromatic ring impaired the ability to block TPC2. The diaryl or benzyl ethers were necessary for the inhibitory effect as well as a basic nitrogen atom. Tertiary amines were preferred because secondary amines caused unspecific effects in calcium imaging experiments. These results further demonstrated that whole endoysosomal patch clamp experiments are still state-of-the-art for ultimate analysis while calcium imaging experiments can be used for fast ligand identification.

77 RESULTS AND DISCUSSION

3.6.3 TPC2 and cancer

The influence of TPC2 functions on cancer hallmarks is of high interest[34] which made TPC2 an interesting target for the development of novel anticancer therapeutics. Hence a close cooperation with Martin Müller (group of Prof. Dr. Angelika Vollmar, LMU) was initiated to use small-molecule inhibitors of TPC2 to further study the role of TPC2 in cancer. All experiments discussed in this chapter were performed by Martin Müller and are to be published in a corporate publication.

Thus, after the identification of the novel TPC2 inhibitors, their potential to inhibit cancer cell growth was investigated. More negative controls (SG-089 (181), SG-121 (185), SG-121-NP (189)) and substances of the Z series were also screened for their antiproliferative properties using RIL175 cells in a CTB assay. Numerous molecules with an increased potency in comparison to tetrandrine (1, IC50: 9.1 µM, entry 1, Table 13) were identified, including truncated variants with IC50 values in the low micromolar range. Monomeric benzyltetrahydroisoquinolines bearing additional aromatic residues (phenyl or benzyl ethers) at both benzenoid rings were found to have outstanding properties. When modifying the amino group, antiproliferative activity remained in a similar range. N-Alkyl residues of different lengths with a basic nitrogen were equipotent (NH (Z3 (137)); N-methyl (SG-005 (187), SG-159 (206));

N-ethyl (SG-158 (208)), IC50: 2.4-4.8 µM, entry 2, 17-18, 28, Table 13). N-Acyl variants lost their antiproliferative properties as well as their basicity (SG-089 (181), SG-145 (183), IC50: ≥ 33 µM, entry 12, 25, Table 13). Variations at C-6 and C-7 of the isoquinoline unit had a slight effect. In most cases, the loss of the aromatic substituents (aryl- or benzyl ether) in C-7 position slightly decreased antiproliferative activity (SG-083 (188), SG-127 (194), SG-157 (195), IC50: 7.6-11 µM, entry 8, 10, 16, Table 13). The same was observed if the 1-benzyl group was replaced by a phenyl group (SG-122 (193) vs. SG-157 (195), entry 9, 16, Table 13), whereas shifting a benzyloxy residue from position C-7 to C-6 had no influence (Z3 (137) vs. Z5 (144), entry 28-29, Table 13). Furthermore, the impact of modifications of the 1-benzyl residue was investigated. Substitution patterns, mostly differing in meta- or para-position, did not markedly affect antiproliferative activity (SG-005 (187), SG-159 (206), Z6 (145), Z11 (141), Z13 (150),

Z15 (152), Z18 (154), Z20 (155), IC50: 1.2-4.8 µM, entry 2, 18, 30-36, Table 13). Miscellaneous diaryl ethers at the 1-benzyl residue were synthesized bearing both electron-donating and - releasing substituents, mainly in meta- or para-position (SG-153 (199), SG-154 (198), SG-155 (196), SG-161 (197), SG-162 (200), SG-164 (201), SG-165 (202)). In most cases, no changes in antiproliferative potencies were observed (IC50: 3.9-5.6 µM, entry 13-15, 19-23, Table 13).

However, cyano and chlorine substituents slightly (SG-164 (201), SG-165 (202), IC50: 8.5-9.1 µM, entry 22-23, Table 13) and the pyridine moiety strongly reduced the antiproliferative activity (SG-163 (203), IC50: 22 µM, entry 21, Table 13). The loss of both, benzyl or diaryl ether moieties resulted in significant reduction (SG-132 (159), IC50: 11 µM,

78 RESULTS AND DISCUSSION entry 11, Table 13) or complete loss of activity (SG-089 (181), SG-121 (185), SG-121-NP

(189), IC50: > 50 µM, entry 25-27, Table 13).

Table 13: Screening of the benzyltetrahydroisoquinoline compound library by cell proliferation assays. Antiproliferative effects of the respective compounds against RIL175 WT cells were assessed by ® CellTiter-Blue cell viability assays. Cells were treated for 72 h with the indicated concentrations. IC50 values were calculated by nonlinear regression. Experiments were performed by Martin Müller.

entry compound IC50 (RIL157) entry compound IC50 (RIL157) 1 tetrandrine (1) 9.1 µM 25 SG-089 (181) >50 µM 2 SG-005 (187) 2.4 µM 26 SG-121 (185) >50 µM 3 SG-094 (192) 3.7 µM 27 SG-121-NP (189) >50 µM 4 berbamine HCl (10) 7.7 µM 28 Z3 (137) 2.9 µM 5 cepharanthine (140) 6.8 µM 29 Z5 (144) 2.7 µM 6 oxyacanthine sulfate (138) 11 µM 30 Z6 (145) 2.9 µM 7 dauricine (11) 9.3 µM 31 Z11 (148) 1.2 µM 8 SG-083 (188) 7.6 µM 32 Z13 (150) 3.5 µM 9 SG-122 (193) 4.5 µM 33 Z15 (152) 2.7 µM 10 SG-127 (194) 8.0 µM 34 Z16 (153) 2.6 µM 11 SG-132 (159) 11 µM 35 Z18 (154) 4.1 µM 12 SG-145 (183) 33 µM 36 Z20 (155) 4.2 µM 13 SG-153 (199) 4.9 µM 14 SG-154 (198) 3.9 µM 15 SG-155 (196) 4.4 µM 16 SG-157 (195) 11 µM 17 SG-158 (208) 4.8 µM 18 SG-159 (206) 4.2 µM 19 SG-161 (197) 4.5 µM 20 SG-162 (200) 5.6 µM 21 SG-163 (203) 32 µM 22 SG-164 (201) 9.1 µM 23 SG-165 (202) 8.5 µM 24 Z22 (157) 18 µM

All benzyltetrahydroisoquinolines, which carry two aryl or benzyl ether groups and a basic amine, inhibited proliferation of RIL175 cells to a similar extent or stronger than tetrandrine (1). The two simplest benzyltetrahydroisoquinolines of this type, SG-005 (187) and SG-094 (192), were subjected to further investigations. SG-005 (187, IC50: 2.4 µM) and SG-094 (192, IC50: 3.7 µM) both displayed markedly enhanced antiproliferative effects, compared to tetrandrine (1). SG-005 (187) and SG-094 (192) had similar or increased antiproliferative potencies against various other cancer cell lines, including human hepatocellular carcinoma (HepG2), human colorectal adenocarcinoma (HCT-15) and human vincristine-resistant acute lymphoblastic leukemia (VCR-R CEM) (entry 1-9, Table 14). While comparing IC50 values of the cell proliferation assay and Fluo-4 based calcium imaging, it was striking that not all compounds that block proliferation were TPC2 inhibitors (SG-132 (159), entry 12, Table 12, entry 11, Table 13) and not all TPC2 inhibitors blocked proliferation (SG-163 (203), entry 22,

79 RESULTS AND DISCUSSION

Table 12, entry 21, Table 13). This indicated that these compounds have side effects on further targets in cancer cells.

Table 14: Antiproliferative effects of tetrandrine (1), SG-005 (187) and SG-094 (192) on different cancer cell lines. Antiproliferative effects of the respective compounds on HCT-15, HepG2 and VCR-R CEM cells were assessed by CellTiter-Blue® cell viability assays. HCT-15 and HepG2 cells were treated for 72 h, VCR-R CEM cells were treated for 48 h. IC50 values were calculated by nonlinear regression. Experiments were performed by Martin Müller.

entry compound cell line IC50 (Ril-175) 1 tetrandrine (1) HCT-15 9.7 µM 2 tetrandrine (1) HepG2 9.4 µM 3 tetrandrine (1) VCR-R CEM 15 µM 4 SG-005 (187) HCT-15 7.8 µM 5 SG-005 (187) HepG2 7.8 µM 6 SG-005 (187) VCR-R CEM 5.5 µM 7 SG-094 (192) HCT-15 7.6 µM 8 SG-094 (192) HepG2 9.9 µM 9 SG-094 (192) VCR-R CEM 10 µM

Cellular uptake, toxicity and the effects on angiogenesis and glucose metabolism of SG-005 (187) and SG-094 (192) were investigated, all in comparison to tetrandrine (1). SG-132 (159) and SG-145 (183) both identified as non-TPC2 blockers by Ca2+ imaging experiments were used as controls. The vascular endothelial growth factor (VEGF) has been demonstrated to be a major contributor to angiogenesis. The role of TPC2-mediated Ca2+ release in neoangiogenesis was already described in literature[35, 52] and VEGF inhibitors were used to treat cancer[38]. Western blot analysis revealed that SG-005 (187) and SG-094 (192) were capable of significantly reducing VEGF-induced phosphorylation levels, while tetrandrine (1) had no effect under the chosen treatment conditions. As expected, the analysis of the selected controls, SG-132 (159) and SG-145 (183), showed no reduced phosphorylation of the investigated VEGFR2 downstream targets.

As the inhibitory effect on TPC2 was similar for tetrandrine (1) and SG-005 (187), the question arose if they differ in their pharmacokinetic properties. Therefore, together with Dr. Christoph Müller and Anna Niedrig, the cellular uptake was investigated by analytical HPLC. Two different cell lines (HUVECs and RIL175 cells) at two different compound concentrations (2 µM and 10 µM) were tested. Incorporation of SG-005 (187) into HUVECs was slightly higher than the uptake of tetrandrine (1) and SG-094 (192) at 2 µM, while all other settings did not show significant differences. In general, cellular uptake for the cancerous liver cells (RIL175) was preferred compared to the primary endothelial cells (HUVECs). Initial uptake was not a major trigger for the improved on-target activity of SG-005 (187) and SG-094 (192) on TPC2.

Cancer cells tend to reprogram their energy metabolism in order to boost extensive cell division[38] and according to the Warburg effect, many cancer cells preferentially use glycolysis

80 RESULTS AND DISCUSSION for ATP production[170]. Normal cells, however, favor oxidative phosphorylation and conduct glycolysis to generate pyruvate out of glucose for the dismantling of unnecessary biomass. Glucose metabolism was identified as one pathway that is altered as a result of TPC2 deficiency. Upon loss of TPC2 function or pharmacological inhibition of this channel with tetrandrine (1), SG-005 (187) and SG-094 (192), a metabolic shift towards a less glycolytic, and therefore healthier, phenotype was observed.

The toxicity of tetrandrine (1) is a serious drawback, especially for in vivo studies. It is postulated to be related to metabolic activation by cytochrome P450 (CYP) enzymes[70, 171] or mitochondrial pathways[172]. Both, SG-005 (187) and SG-094 (192) showed decreased toxicity to non-cancerous hepatic stem cells (HepaRGTM) and peripheral blood mononuclear cells (PBMCs), which was not related to CYP3A4 levels. This indicates that metabolic oxidation by CYP enzymes is not responsible for the toxicity of tetrandrine (1). Thus, truncation of tetrandrine (1) slightly (SG-005 (187)) or substantially (SG-094 (192)) improved the toxicity prolife.

In vivo studies using tetrandrine (1) are limited due to the poor solubility as well as the high toxicity. The simplified, less toxic and good soluble substances SG-005 (187) and SG-094 (192) were evaluated for their therapeutic potential in an ectopic mouse model using C57Bl/6- Tyr mice and RIL175 HCC cells. SG-005 (187) did not show the expected in vivo efficacy, while SG-094 (192), successfully managed to reduce tumor growth in vivo at tolerated doses.

In short, pharmacological inhibition of TPC2 function reduced cancer cell proliferation, altered cellular energy metabolism and prevented tumor growth. The two small-molecule TPC2 inhibitors, SG-005 (187) and SG-094 (192), showed a great drug-likeness compared to the limited panel of currently available direct TPC2 blockers. The cellular uptake could be improved and the toxicity was lowered in comparison to tetrandrine (1). Using these molecules enables to perform in vivo experiments which increases their possible applications.

81 RESULTS AND DISCUSSION

3.6.4 Separation of the enantiomers

The racemic mixtures of SG-005 (187) and SG-094 (192) were separated by semipreparative chiral HPLC in order to identify the eutomer. The absolute stereochemistry was determined using ECD spectra and was confirmed by computational calculations by Aaron Gerwien (group of Prof. Dr. Henry Dube, LMU) (Figure 26 C and F). The separated enantiomers were analyzed in Fluo-4 based calcium imaging and compared with the racemic mixtures, respectively. For activation of the ion channel both TPC2 activators, TPC2-A1-N (16) and TPC2-A1-P (17) were used. (S)-SG-005 (209) showed a decreased potency (IC50: 7.8 µM) compared to its enantiomer (R)-SG-005 (210, IC50: 2.4 µM) after activation with TPC2-A1-N (16, Figure 26 E), while there was no difference after activation with TPC2-A1-P (17, Figure 26 D). The same applied to (S)-SG-094 (211, IC50: 31 µM) and (R)-SG-094 (212, IC50: 14 µM) to some extent (Figure 26 A and B). Thus, all enantiomers showed inhibition of TPC2 after activation with both activators.

Figure 26: Fluo-4 based calcium imaging of enantiopure SG-005 (187) and SG-094 (192). (A-B) Concentration-effect relationships using HEK293 cells stably expressing hTPC2L11A/L12A-RFP. Cells were sequentially stimulated with SG-094 (rac: 192, (S): 211, (R): 212) and TPC2-A1-P (17, 10 µM, A) or TPC2-A1-N (16, 10 µM, B) as activator. IC50 values were calculated out of at least three independent experiments using GraphPad. (C) Structures of both enantiomers of SG-094 ((S): 211, (R): 212). (D-E) Experiment as in A-B but using SG-005 (rac: 187, (S): 209, (R): 210). (F) Structures of both enantiomers of SG-005 ((S): 209, (R): 210). Experiments were performed by Nicole Urban.

The different enantiomers were further analyzed for toxicity and their ability to inhibit proliferation. All compounds were not toxic to non-cancerous HepaRG cells in a CTB assay at

82 RESULTS AND DISCUSSION

10 µM, while tetrandrine (1) already showed toxicity at this concentration (Figure 27 A). In particular, SG-094 (192) and enantiomers (211, 212) showed a significantly decreased toxicity compared to tetrandrine (1). Antiproliferative properties of these compounds were evaluated using RIL175 cells in a CTB assay. Both enantiomers of both compounds did reduce cell proliferation, though both (R)-enantiomers (210, 212) were less potent than the corresponding (S)-enantiomers (209, 211) and the racemates (187, 192), as depicted in Figure 27 B and C.

Figure 27: Toxicity and cell proliferation of the enantiopure compounds. (A) Toxicity of tetrandrine (1), SG-005 (rac: 187, (S): 209, (R): 210) and SG-094 (rac: 192, (S): 211, (R): 212) to non-cancerous cells was probed by treating HepaRG cells with 10 µM of the corresponding compound for 72 h. Cell viability was assessed by CellTiter-Blue® cell viability assay and was normalized to vehicle control. Bar graphs indicate mean ± SEM of three independent experiments. **p < 0.01, *p < 0.05, using one-way ANOVA followed by Tukey’s post hoc test. (B-C) Antiproliferative effects of the SG-005 (B, rac: 187, (S): 209, (R): 210) and SG-094 (C, rac: 192, (S): 211, (R): 212) were assessed by CellTiter-Blue® cell viability assays. RIL-175 cells were treated for 72 h. IC50 values were calculated by nonlinear regression. Fluorescence intensities were normalized to vehicle control and are displayed as mean ± SEM of three independent experiments. Experiments were performed by Martin Müller.

Notably, calcium imaging revealed that both enantiomers of SG-005 (187) and SG-094 (192) inhibit TPC2 upon activation to a similar extent. Furthermore, the racemates showed virtually the same effects as the more potent compound in cell proliferation assay. Consequently, the racemates were preferred for all subsequent experiments.

3.6.5 Expanding the TPC2 inhibitor panel The aforementioned panel of TPC2 inhibitors comprises indirect TPC inhibitors like trans-Ned- 19 (2)[36, 60, 173] and direct inhibitors like the flavonoid naringenin (8)[52], the listed drugs fluphenazine (6) and raloxifene (7)[31] and the bisbenzylisoquinoline alkaloid tetrandrine (1)[2, 36]. There are other reported compounds which interfere with NAADP-mediated Ca2+ signaling like BZ194, an N-alkylated nicotinic acid derivative[174], and pyridoxal phosphate 6-azophenyl- 2′,4′-disulfonic acid (PPADS)[175, 176]. For both compounds, however, no direct measurements of TPC inhibition are published, questioning the accuracy of these inhibitors.

Naringenin (8) and trans-Ned-19 (2) require high concentrations to inhibit TPCs (500 µM and

83 RESULTS AND DISCUSSION

[52, 60] 125 µM, respectively) . Raloxifene (7) and fluphenazine (6) both exhibite a low IC50 in endo-lysosomal patch clamp experiments (0.63 µM and 8.2 µM) after stimulation with PI(3,5)P2 (4)[31]. The application of raloxifene (7), however, is limited to postmenopausal women regarding the influence upon hormone levels and fluphenazine (6) is known for its broad spectrum of adverse effects. The most prominent and potent TPC inhibitor, tetrandrine (1), blocks TPCs at nano-molar concentrations (500 nM) in endo-lysosomal patch clamp experiments[2]. It is used in different studies as exemplary TPC inhibitor, but the poor solubility combined with the reported toxicity make tetrandrine (1) not applicable for in vivo studies. None of these antagonists of TPCs is selective, neither can they discriminate between the two TPC isoforms.

The new developed TPC2 inhibitors, SG-005 (187) and SG-094 (192) showed improved efficacy in electrophysiological endo-lysosomal patch clamp experiments for SG-094 (192, 74% inhibition) or equipotent efficacy for SG-005 (187, 44%), compared to tetrandrine (1, 54%) after activation with PI(3,5)P2 (4). These results were confirmed by Fura-2 calcium imaging experiments. Again SG-094 (192) had the strongest effect (**) compared to tetrandrine (1, *) and SG-005 (187, *) when applied at a concentration of 10 µM. Furthermore the cellular uptake could be improved and the toxicity was lowered in comparison to tetrandrine (1). The great solubility of SG-005 (187) and SG-094 (192) enabled in vivo studies in an ectopic mouse model. Both compounds were well tolerated at given doses and SG-094 (192), successfully managed to reduce tumor growth in vivo. These results demonstrate that the two small- molecule TPC2 inhibitors, SG-005 (187) and SG-094 (192), showed a promising drug-likeness and expanded the limited panel of available TPC2 blockers.

84 RESULTS AND DISCUSSION

3.7 Side Projects

The developed calcium imaging methods were further used in some side projects to confirm inhibitory effects on TPC2 or TRPML channels. Within projects in the group of Prof. Dr. Bracher substances were synthesized or purchased and subsequently tested by me on TPC2 to evaluate their effect on endo-lysosomal ion channels.

3.7.1 The isoquinoline-benzylisoquinoline alkaloid rac-muraricine (213)

Ramona Schütz performed the first racemic total synthesis of the isoquinoline- benzylisoquinoline alkaloid rac-muraricine (213, Figure 28 A)[177]. Pharmacological characterization identified rac-muraricine (213) as a moderate inhibitor of P-glycoprotein, while it showed only low antiproliferative effects. Structural similarities to tetrandrine (1) and dauricine (11) led to the presumption that rac-muraricine (213) can block TPC2. Therefore Fura-2 based calcium imaging was performed using TPC2-A1-N (16) for activation. The alkaloid rac-muraricine (213) was not able to inhibit TPC2 activation in comparison to a DMSO control (Figure 28 B and C).

Figure 28: Fura-2 based calcium imaging results of rac‐muraricine (213) and a DMSO control using the TPC2 activator TPC2‐A1‐N (16). (A) Structure of rac‐muraricine (213). (B) Statistical analysis of the maximal change in the Fura‐2 ratio (mean ± SEM) with the number of independent experiments in parentheses. An unpaired t ‐test was applied. ns = not significant. (C) Representative Ca2+ signals recorded from HEK293 cells stably expressing TPC2L11A/L12A–RFP. After applying rac‐muraricine (213, 10 µM; n = 19 single cells) or DMSO (0.1% in HBS; n = 16 single cells) and monitoring the signal for 400 s, cells were stimulated with TPC2‐A1‐N (16, 10 µM) and further recorded for 400 s. Highlighted lines represent the mean response from a population of cells. Shaded traces represent responses of single cells. All experiments were performed on a Leica DMi8 live cell microscope.

85 RESULTS AND DISCUSSION

3.7.2 The TRPML inhibitors ML-SI1 (214) and ML-SI3 (215)

In the course of an Erasmus project the TRPML inhibitor ML-SI1 (214) was synthesized under the supervision of Dr. Marco Keller (Figure 29 D). This compound was one out of two published TRPML inhibitors with undefined stereochemistry[178]. The product was received as a racemic mixture of two diastereomers (4 isomers in total) and was tested on the TRPML channels for its postulated inhibitory effect. Experiments were performed by first activating the ion channel with ML-SA1 or MK6-83 (10 µM, respectively) and subsequent inhibition using ML-SI1 (214, 10 µM) or ML-SI3 (214, 10 µM). For statistical analysis the median activation was normalized to 1 to compare the percentage inhibitory effect.

Figure 29: Fura-2 based calcium imaging experiments for TRPML inhibitors. (A) Statistical analysis of the inhibitory effect of ML-SI1 (214) on TRPMLs in Fura-2 based Ca2+ imaging experiments (normalized activation). Experiments were carried out as previously described[115] on a Polychrome IV mono- chromator (for hTRPML1) or a Leica DMi8 live cell microscope (for TRPML2 and 3). After stimulation with ML-SA1 (10 µM) for 200 s, the inhibitor ML-SI1 (214, 10 µM) was applied for another 200 s. For measurements HEK293 cells stably expressing hTRPML2-YFP or hTRPML3-YFP, and transiently transfected hTRPML1ΔNC-YFP cells were used[114]. (B) Statistical analysis as in (A), using ML-SA1 (10 µM) or MK6-83 (10 µM) for activation of hTRPML1ΔNC-YFP transiently transfected HEK293 cells. (C) Representative Ca2+ signals recorded from hTRPML1ΔNC-YFP transiently transfected HEK293 cells, loaded with Fura-2/AM (126) and sequentially stimulated with ML-SA1 (10 µM, red, n = 5 transfected and 2 NT cells) or MK6-83 (10 µM, orange, n = 11 transfected and 3 NT cells) and ML-SI1 (214, 10 µM). Highlighted lines represent the mean response from a population of cells. Shaded traces represent responses of single cells. (D) Structure of rac-ML-SI1 (214), synthesized by Dr. Marco Keller. (E)

86 RESULTS AND DISCUSSION

Statistical analysis of the inhibitory effect of ML-SI3 (215) as in (A) using ML-SA1 (10 µM) for activation and ML-SI3 (215, 10 µM from Enamine store) for inhibition. All experiments were performed on a Leica DMi8 live cell microscope. (F) Statistical analysis as in (B) but inhibition with ML-SI3 (215, 10 µM). All experiments were performed on a Leica DMi8 live cell microscope. (G) Representative Ca2+ signals as in (C) but performed on a Leica DMi8 live cell microscope. Cells were sequentially stimulated with ML- SA1 (10 µM, blue, n = 5 transfected and 8 NT cells) or MK6-83 (10 µM, green, n = 4 transfected and 5 NT cells) and ML-SI3 (215, 10 µM). Highlighted lines represent the mean response from a population of cells. Shaded traces represent responses of single cells. In all statistical analyses of Ca2+ imaging experiments, mean values of n independent experiments are shown as indicated. ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant, one-way ANOVA test followed by Tukey’s post-hoc test.

Single cell calcium imaging experiments confirmed that the synthesized racemic ML-SI1 (214) had an inhibitory effect on TRPML1 (Figure 29 A-C). Statistical analysis of the three isoforms of the ion channel showed strong inhibitory effect on hTRPML1, a weaker effect on hTRPML2 and no effect on hTRPML3, all after activation with ML-SA1 (Figure 29 A). While inhibition after activation with ML-SA1 showed a robust signal, it was not possible to significantly block MK6-83 induced activation (Figure 29 B and C). This indicates an activator dependent inhibition.

The second published inhibitor, ML-SI3 (215)[178], was commercially available at Enamine store (CAS: 891016-02-7) but with undefined stereochemistry. Within a new project, Charlotte Leser identified the commercially available ML-SI3 (215) as racemic mixture of trans-ML-SI3 (215, Figure 29 H). The commercially available ML-SI3 (215, Figure 29 H) was able to block ML- SA1 evoked hTRPML1 and 2 activation around 50% but did not inhibit hTRPML3 activation (Figure 29 E). The comparison of the different activators showed that ML-SI3 (215) was able to block both ML-SA1 and MK6-83 induced activation in the same manner (Figure 29 F and G). Thus, ML-SI3 (215) seemed the more promising antagonist for further investigations.

87 SUMMARY

4 Summary

The first aim of this thesis was the identification of small-molecule TPC2 activators. This was achieved by performing a high-throughput screening of a compound library, containing 80000 substances. Two TPC2 activators (TPC2-A1-N (16), TPC2-A1-P (17)) were identified within this screening process. Both compounds were confirmed by fluorescence-based Ca2+-imaging and electrophysiological endo-lysosomal patch clamp measurements. To verify the structures, both substances were synthesized and re-evaluated.

Sjogren et al. published a two-step synthesis for α-cyano-β-hydroxypropenamides, which resulted in the desired TPC2-A1-N (16)[91]. Only slight changes in this procedure were necessary for optimization. Different aniline building blocks (IV, Figure 30 A), 2-cyanoacetic acid (18) and DCC as coupling reagent yielded 23 cyanoacetanilides (II, Figure 30 A), nearly all in high yield. Acylation of these intermediates (II, Figure 30 A) using different benzoic acid building blocks (III, Figure 30 A) and NaH as a strong base gave the desired α-cyano-β-hydroxypropenamides (I, Figure 30 A) as analogs of TPC2-A1-N (16, Figure 30 B). Only the thio-analog SGA-167 (91) was synthesized with another method. Collectively, 47 compounds were synthesized in moderate to high yield, including the hit TPC2-A1-N (16), and two drugs (prinomide (58), teriflunomide (76)).

Fura-2 based calcium imaging experiments were performed to study SAR. Surprisingly, none of the 46 modified versions of TPC2-A1-N (16, Figure 30 B) did show significantly increased efficacies. Replacing the p-trifluoromethyl group on the anilide side of the molecule (R1; blue) with other electron-withdrawing groups in para-position did not lead to significant changes. The introduction of electron-releasing groups in para-position was tolerated to some extent (SGA- 4 (47), SGA-84 (72)). Meta-disubstitution patterns were most beneficial for the benzoyl ring system (R2, green), while more drastic changes in this aromatic region caused a complete loss of activity. This was demonstrated for the approved drugs teriflunomide (76) and prinomide (58) as well as the 4-trifluoromethyl variant of prinomide (SGA-32 (59)), all possessing methyl or pyrrole residues instead of the benzoyl ring. In total, 24 analogs were able to activate TPC2 with similar or lower efficacy compared to TPC2-A1-N (16)[22, 135]. All results were confirmed by determination of concentration-effect relationships, underlining the very flat SAR of this chemotype on TPC2. The increased potency and efficacy of SGA-85 (73) were relativized by non-specific effects on control cells. Selectivity-screening revealed that SGA-108 (77) showed an improved selectivity spectrum, which explains the loss of efficacy. All these findings have proven the high efficacy of TPC2-A1-N (16) and the fact that it is the most promising candidate to be a great tool compound.

88 SUMMARY

Figure 30: Synthesis and compound overview of TPC2-A1-N (16) and analogs. (A) Synthesis scheme for the preparation of most α-cyano-β-hydroxypropenamides. (B) Structures of TPC2-A1-N (16) and analogs. Differences to the hit compound are marked in magenta (TPC2 activators) or red (no TPC2 activators).

The synthesis of the second activator, TPC2-A1-P (17), was more challenging than the synthesis of TPC2-A1-N (16), as the required intermediate, an α-halogenated acetophenone (IV, Figure 31) substituted in C-2 position with a trifluoromethoxy and in C-5 position with a bromo group, was not commercially available. Many different synthetic approaches were not successful until the main problem was detected: The desired product was highly volatile.

89 SUMMARY

Hence, a synthesis avoiding these conditions was chosen and the α-halogenated acetophenone (IV, Figure 31 A) was synthesized successfully.

For the synthesis of the highly substituted TPC2-A1-P scaffold a Paal-Knorr pyrrole synthesis was performed. Under alkaline conditions 1,4-diketones (VIII, Figure 31 A) were synthesized, using the α-halogenated acetophenone (IV, Figure 31 A) and β-ketoester (V, Figure 31 A) building blocks. These 1,4-diketones (VIII, Figure 31 A) were not isolated and directly condensed with a primary amine building block (III, Figure 31 A) in acidic medium to give multi-substituted pyrroles (II, Figure 31 A). In total, 13 esters were synthesized and 8 of them isolated. Hydrolysis of the esters (II, Figure 31 A) required harsh conditions, utilizing microwave irradiation for several hours to give the hit TPC2-A1-P (17) and 12 analogs varying all substituents of the pyrrole moiety (Figure 31 B).

Figure 31: Synthesis and compound overview of TPC2-A1-P (17) and analogs. (A) Synthesis scheme for the synthesis of TPC2-A1-P (17) and analogs. (B) Structures of TPC2-A1-P (17) and analogs. Differences to the hit compound are marked in magenta (TPC2 activators) or red (no TPC2 activators).

Similar to TPC2-A1-N (16), calcium imaging experiments were performed to gain first insights into SAR. Again, all modified versions of TPC2-A1-P (17) showed no improvement of efficacy[22]. Every change in structure resulted in a decrease or total loss of activity. SAR revealed that the free carboxylic acid is essential for the activating effect, as the ester SGA- 140 (109) was not active. It could possibly serves as a prodrug of TPC-A1-P (17) in living systems, but this has not been investigated yet. Both substituents at the phenyl ring, the trifluoromethoxy and the bromine group, were essential for activating TPC2, as exemplified by the inactive methoxy (SGA-55, 115) and des-bromo (SGA-162, 120) analogs. Moderate expansion of the size of the pyrrole substituent at C-2 position (methyl in TPC2-A1-P (17) vs. ethyl in SGA-152 (121) and phenyl analog SGA-154 (122)), had the same effect. The only fairly tolerated structure modification was replacing the cyclohexylmethyl moiety in 1-positon

90 SUMMARY of the pyrrole ring by a benzyl residue (SGA-150, 125), whereas linear or branched alkyl chains (SGA-149 (123), SGA-153 (124)) caused loss of activity (Figure 31 B). All results were further confirmed by full concentration-effect relationships, generated by Fluo-4 calcium imaging experiments. These harsh limitations showed that TPC2-A1-P (17) had an unusually steep structure-activity relationship resulting in TPC2-A1-P (17) as great chemical tool.

Comparing the results of both activator classes, a considerable number of TPC2-A1-N (16) analogs caused remarkable TPC2 activation, but none of them showed significantly increased efficacies. In contrast to TPC2-A1-N (16), TPC2-A1-P (17) showed an unusually steep structure-activity relationship, and except for one analog (SGA-150, 125) none of the analogs showed any activity. TPC2-A1-N (16) itself and some of its analogs bearing residues in para- position at the benzoyl ring were known as anthelmintic agents[91], while TPC2-A1-P (17) was only namely mentioned as a precursor in the synthesis of cannabinoid-1 receptor (CB1R) inverse agonists[105]. Two analogs of TPC2-A1-N (16) were launched drugs (teriflunomide (76), prinomide (58)), proving the drug-likeness of the TPC2-A1-N skeleton. There were no drugs comprising the TPC2-A1-P skeleton and furthermore no reports for using the free carboxylic acids. Thus, the two HTS hits TPC2-A1-N (16) and TPC2-A1-P (17) can be regarded as novel, strong chemical tools.

In a very recent publication we published initial steps for the pharmacological profile of TPC2- A1-N (16) and TPC2-A1-P (17)[22]. The subtype selectivity to TPC1 and cell permeability were proven. These small-molecules finally helped to resolve the conflict, whether TPC2 is a

2+ [5, 12, 14, 127, 128] + [15, 20, 129] NAADP-activated Ca release channel or a PI(3,5)P2 gated Na channel : TPC2 is a ligand-dependent, non-selective cation channel with malleable cation selectivity. Depending on the agonist, TPC2 shows higher calcium permeability (activation with TPC2-A1- N (16), similar to NAADP (3)) or increased sodium permeability (activation with TPC2-A1-P

[8] (17), similar to PI(3,5)P2 (4)). Additionally the PI(3,5)P2 (4) binding site in TPC2 has been identified to be the overlapping with the pore for TPC2-A1-P (17). While TPC2-A1-N (16) induced an alkalinization of single vesicles in cells expressing wild-type TPC2, as described for NAADP (3)[130, 131], TPC2-A1-P (17) promoted lysosomal exocytosis.

In comparison with other new TPC2 activators, identified by Zhang et al.[111], our two new activators were more potent and selective, which made them the perfect chemical tools for the identification of TPC2 inhibitors.

Because of their anti-cancer or anti-viral effects TPC2 inhibitors are currently of higher therapeutic significance than activators. Hence, a set of TPC2 inhibitors was synthesized, inspired by the known TPC2 inhibitor tetrandrine (1). These molecules mimicked one half of the bisbenzylisoquinoline tetrandrine (1) and further decoration, not only in form of O-benzyl derivatives but also as diaryl ethers, was supposed to closely imitate tetrandrine (1) in shape

91 SUMMARY and size (Scheme 34 A). Iturriaga-Vásquez et al. designed simplified and more accessible congeners of tetrandrine (1) bearing extensions as benzyl groups, which were developed as L-type calcium channel blockers[82]. This was a promising hint for the plan to design truncated analogs of tetrandrine (1).

Starting materials for the synthesis of the desired tetrahydroisoquinolines were carbamates and enol ethers (I and II, Scheme 34 B). Using ethyl chloroformate (162), NEt3 and 3- methoxytyramine (160) or its O-protected derivatives gave different carbamates (I). Enol ethers (II) were received via Wittig olefination of commercially available para-substituted benzaldehydes. These precursors underwent N-acyl Pictet-Spengler reaction using TFA and to yield N-acethoxycarbonyl-1-benzyltetrahydroisoquinolines (III). Tertiary N-methyl amines (IV) were received by reduction of the carbamate group and, if applicable, in situ deprotection of carbonate-protected phenols using lithium alanate. Diverse diaryl ethers (V) were introduced at the generated phenolic groups via gentle Chan-Evans-Lam coupling with the appropriate arylboronic acids. Furthermore, three routes were established to yield SG-005 (187): lithium alanate reduction of the carbamate SG-145 (183), Eschweiler-Clarke reaction starting with the secondary amine Z3 (137) and Mitsunobu reaction on N-methylcoclaurine (SG-132, 159) to receive benzyl protecting groups. The Eschweiler-Clarke reaction using Z6 (145) also yielded SG-159 (206) and an alkylation reaction using bromoethane yielded SG-158 (208).

The racemates SG-005 (187) and SG-094 (192) were separated by semi-preparative chiral HPLC for the identification of the potential eutomers. The absolute configuration of the enantiomers was determined using ECD spectra and confirmed by computational calculations performed by Aaron Gerwien. Notably, Ca2+ imaging revealed that both enantiomers of SG- 005 (187) and SG-094 (192) inhibit TPC2 upon activation to a similar extent. Consequently, the racemates were used for all further experiments.

A Fura-2 based single cell calcium imaging method was developed and the TPC2-blocking effect of SG-005 (187) and SG-094 (192) was confirmed. These two substances were highlighted within this project because of their outstanding effects in a variety of experiments. While tetrandrine (1) and SG-005 (187) were equipotent (54% and 44% inhibition or *, respectively), SG-094 (192) was significantly more potent (74%, **) at the tested conditions

(10 µM of inhibitor, activated with 1 µM PI(3,5)P2 (4)) in endo-lysosomal patch-clamp experiments (performed by Dr. Yu-Kai Chao) and Fura-2 based calcium imaging experiments (10 µM TPC2-A1-N (16), followed by 10 µM of the respective inhibitor). Furthermore, two negative controls were identified to assure the accuracy of the experiments. SG-132 (159, N- methylcoclaurine) is missing the decoration on the phenol groups and SG-145 (183) the alkaline nitrogen function of the tetrahydroisoquinoline, which represent the two most important structural moieties.

92 SUMMARY

Scheme 34: Summary for the synthesis of truncated tetrandrine analogs. (A) Schematic overview for the truncation of tetrandrine (1). Decorations on the former phenols (R’s) make the molecule resemble tetrandrine (1) even more. (B) Complete synthesis branches for the synthesis of truncated analogs, starting with carbamate and enol ether building blocks. N-acyl Pictet-Spengler reaction resulted in benzyltetrahydroisoquinolines, that were deprotected and reduced using lithium alanate. The resulting tertiary amines received their final decoration using the Chan-Evans-Lam coupling. Shaded reactions are additional reactions to receive different benzylated analogs.

All compounds were analyzed for their ability to inhibit TPC2-A1-N (16) and TPC2-A1-P (17) induced TPC2 activation in Fluo-4 calcium imaging experiments performed by Nicole Urban.

Within this screening IC50 values were obtained not only for the synthesized substances, but also for more alkaloids: intermediates of the morphine biosynthesis like racemic coclaurine (Z1, 141), kindly provided by Prof. Dr. Meinhart Zenk†, tetrandrine (1) and derivatives as fangchinoline (9), kindly provided by Prof. Dr. Peter Pachaly†. Commercially available benzylisoquinoline alkaloids like dauricine (11) and oxyacanthine (138) completed the screening library. This screening pointed out that tetrandrine (1), fangchinoline (9) and the other alkaloids inhibit TPC2 to the same extent (around 50%). SG-005 (187), SG-094 (192) and the orientaline derivatives SG-158 (208) and SG-159 (206) were able to inhibit TPC2 activation similarly or even better than tetrandrine (1). None of the further analogs showed

93 SUMMARY significant stronger effects on TPC2. The substances bearing a para-methoxy, para-methyl or their fluorinated variants (SG-153 (199), SG-154 (198), SG-155 (196), SG-162 (200)) showed a lower IC50 value after activation with TPC2-A1-P (17) compared to activation with TPC2-A1- N (16). As mentioned before, SG-005 (187) and SG-094 (192), derived from the alkaloid coclaurine, were selected as most promising TPC2 inhibitors for further investigation.

After the identification of the novel TPC2 inhibitors, their potential to inhibit cancer cell growth was investigated. All 1-benzyltetrahydroisoquinolines carrying two aryl or benzyl ether groups and a basic amine inhibited proliferation of RIL175 cells to a similar or stronger extent than tetrandrine (1). The two simplest 1-benzyltetrahydroisoquinolines, SG-005 (187, IC50: 2.4 µM) and SG-094 (192, IC50: 3.7 µM), both displayed markedly enhanced antiproliferative effects compared to tetrandrine (1) also against various other cancer cell lines including HepG2, HCT-

15 and VCR-R CEM. The comparison of IC50 values of the cell proliferation assay and Fluo-4 based calcium imaging showed that not all compounds that block proliferation were TPC2 inhibitors (SG-132 (159)) and not all TPC2 inhibitors block proliferation (SG-163 (203)), indicating additional effects of these compounds on other targets in cancer cells.

In an effort to conclude the correlation between TPC2 inhibition and hallmarks of cancer, cellular uptake, toxicity and the effects on angiogenesis and glucose metabolism of SG-005 (187) and SG-094 (192) were investigated by Martin Müller (Vollmar group), all in comparison to tetrandrine (1). The non TPC2-blockers SG-132 (159) and SG-145 (183) were used as controls. Pharmacological inhibition of TPC2 function reduced cancer cell proliferation altered cellular energy metabolism and prevented tumor growth in vivo. The cellular uptake could be improved and the toxicity was lowered in comparison to tetrandrine (1). Thus, the two small- molecule TPC2 inhibitors SG-005 (187) and SG-094 (192) showed a great potential for drug development especially considering the limited panel of currently available TPC2 blockers.

Summarizing, big steps to boost the basic research of two pore channels have been achieved within this project. The two novel small-molecule activators of TPC2, TPC2-A1-N (16) and TPC2-A1-P (17), were discovered and analyzed in extensive studies. Now they represent two powerful chemical tools for the analysis of TPC2 function. These activators enable the use of fluorescent based assays on intact cells to study TPC2 inhibitors. Therefore a new generation of TPC2 inhibitors was synthesized, resembling one half of the alkaloid tetrandrine (1) plus the two benzenoid rings of the second benzylisoquinoline moiety. The two new TPC2 inhibitors with improved or at least equipotent ability to inhibit TPC2 were developed, SG-005 (187) and SG-094 (192). Together with our cooperation partners TPC2 was proven to be an exciting target in tumor therapy and these two compounds were identified as promising lead structures for cancer therapy.

94 EXPERIMENTAL PART

5 Experimental part

Synthesis details and analytical data

All chemicals used were of analytical grade and were obtained from abcr (Karlsruhe, Germany), Fischer Scientific (Schwerte, Germany), Sigma-Aldrich (now Merck, Darmstadt, Germany), TCI (Eschborn, Germany) or Th. Geyer (Renningen, Germany). HPLC grade and dry solvents were purchased from VWR (Darmstadt, Germany) or Sigma-Aldrich, all other solvents were purified by distillation. Hydrophobic phase separation filters (MN 617 WA, 125 mm) were purchased from Macherey Nagel (Düren, Germany). All reactions were monitored by thin-layer chromatography (TLC) using pre-coated plastic sheets POLYGRAM® SIL G/UV254 from Macherey-Nagel and detected by irradiation with UV light (254 nm or 366 nm). Furthermore reactions were monitored by atmospheric pressure solids analysis probe (ASAP) via atmospheric-pressure chemical ionization (APCI) on an expressionL CMS device (Advion, Ithaca, USA). Flash column chromatography (FCC) was performed on Merck silica gel Si 60 (0.015 – 0.040 mm). Preparative and semipreparative (chiral) HPLC was performed on a Shimadzu HPLC system consisting of a LC-20AP solvent delivery module, a CTO-20A column oven, a SPD-M20A photodiode array UV/vis detector and a CBM-20A system controller using a semipreparative CHIRALPAK® IC column (particle size 5 μm, Diacel) or a preparative NUCLEODUR® 100-5 column (particle size 5 μm, Macherey-Nagel). Microwave-assisted reactions were carried out in a Discover (S-Class Plus) SP microwave reactor (CEM GmbH, Kamp-Lintfort, Germany). NMR spectra (1H, 13C, DEPT, H-H-COSY, HMQC/HSQC, HMBC) were recorded at 23 °C on an Avance III 400 MHz Bruker BioSpin or Avance III 500 MHz Bruker BioSpin instrument, unless otherwise specified. Chemical shifts δ are stated in parts per million (ppm) and are calibrated using residual protic solvents as an internal reference for proton (CDCl3: δ = 7.26 ppm, (CD3)2SO: δ = 2.50 ppm, C2D2Cl4: δ = 5.91 ppm, CD3OD:

δ = 3.31 ppm, C3D6O: δ = 2.05 ppm) and for carbon the central carbon resonance of the solvent (CDCl3: δ = 77.16 ppm, (CD3)2SO: δ = 39.52 ppm, C2D2Cl4: δ = 74.20 ppm, CD3OD:

δ = 49.00 ppm, C3D6O: δ = 29.84 ppm). Multiplicity is defined as s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. NMR spectra were analyzed with NMR software MestReNova, version 12.0.1-20560 (Mestrelab Research S.L.). High resolution mass spectra were performed by the LMU Mass Spectrometry Service applying a Thermo Finnigan MAT 95 or Joel MStation Sektorfeld instrument at a core temperature of 250 °C and 70 eV for EI or a Thermo Finnigan LTQ FT Ultra Fourier Transform Ion Cyclotron Resonance device at 250 °C for ESI. IR spectra were recorded on a Perkin Elmer FT-IR Paragon 1000 instrument as neat materials. Absorption bands were reported in wave number (cm-1) with ATR PRO450-S. Melting points were determined by the open tube capillary method on a Büchi melting point B- 540 apparatus and are uncorrected. HPLC purities were determined using an HP Agilent 1100 HPLC with a diode array detector and an Agilent Poroshell column (120 EC-C18; 3.0 × 100

95 EXPERIMENTAL PART mm; 2.7 micron) with acetonitrile/water as eluent. Electronic circular dichroism (ECD) spectra were measured on a Jasco J-810 Spectropolarimeter. Optical rotations were further measured at the given temperature (T in [°C]) on a Perkin Elmer 241 Polarimeter instrument using a sodium lamp (Na D-line, 589 nm). Measurements were carried out in a cell with path lengths (l) of 1.0 dm. Concentrations are given in g/100 mL.

96 EXPERIMENTAL PART

5.1 Preparation of TPC2 activators 5.1.1 General procedures

General Procedure A – Synthesis of N-aryl cyanoacetamides

According to Sjogren, et al.[91] the appropriate aniline (1.0 eq.) and 2-cyanoacetic acid (18, 1.0 eq.) were dissolved in DMF and cooled to 0 °C. DCC (1.0 eq.) was added portion wise. The mixture was warmed up to rt over 1 h and subsequently diluted with hexanes/EtOAc (1:1). Precipitates were removed by filtration and the filtrate was extracted once with 1 M aq. HCl and thrice with EtOAc. The combined organic layers were washed with sat. aq. NaCl solution, dried over Na2SO4, filtered and concentrated in vacuo. Recrystallization from EtOH yielded the desired amides.

General Procedure B – Synthesis of α-cyanoaroylacetanilides

According to Sjogren, et al.[91] the appropriate amides received from general procedure A (1.0 eq.) were dissolved in dry THF, the solution was cooled to 0 °C and NaH (dispersion in paraffin, 60%, 2.3 eq.) was added. After stirring for 15 min, the appropriate benzoyl chloride (1.1 eq.) was added. The mixture was stirred at 0 °C for 1 h and then cautiously treated with 1 M HCl. The precipitate was collected by filtration, washed with ice water and cold EtOH and recrystallized from toluene to give the desired cyanoaroylacetanilide.

If the appropriate benzoyl chloride was not commercially available, it was prepared by refluxing the appropriate benzoic acid (1.1 eq.) in SOCl2 (55 eq.) for 1 h and concentrating in vacuo. The resulting acid chloride was immediately transferred to the reaction.

General Procedure C – Paal Knorr Pyrrole synthesis

Following a general procedure published by Kang, et al.[103] the appropriate β-ketoester (1.1 eq.) was dissolved in dry THF and cooled to 0 °C, before NaH (dispersion in paraffin, 60%, 1.5 eq.) was added portion wise. After the suspension was stirred for 30 min, a solution of appropriate halogenated acetophenone (1.0 eq.) and KI (1.0 eq.) in dry THF was added dropwise. The reaction mixture was allowed to warm up to rt over 2 h, then poured on water and extracted thrice with Et2O. The combined organic phases were washed with sat. aq.

NaHCO3 solution, dried over Na2SO4, filtered and concentrated in vacuo. The residue was dissolved in acetic acid and the appropriate primary amine (2.0 eq.) was added dropwise. The reaction mixture was stirred at 80 °C for 18 h. The solvent was removed in vacuo, the residue dispersed in water and extracted thrice with diethyl ether. The collected organic phases were washed with sat. aq. NaHCO3 solution, dried over Na2SO4 and concentrated in vacuo.

97 EXPERIMENTAL PART

Purification was accomplished by FCC and recrystallization from EtOH if not otherwise specified.

General Procedure D – Alkaline deprotection of esters

LiOH (10 eq.) was added to a solution of the appropriate ester (1.0 eq.) in dioxane/H2O (5:1) and the reaction mixture was stirred in a closed vessel under microwave irradiation

(pmax = 8 bar, Pmax = 200 W, Tmax = 180 °C) for 1-18 h. The suspension was diluted with water to thrice original volume and aq. 2 M HCl was added dropwise under vigorous stirring until the mixture was strongly acidic. The formed precipitate was collected by filtration, washed with water and dried. If necessary, the acids were recrystallized from EtOH to yield the pure products.

5.1.2 Synthetic procedures 5.1.2.1 Synthesis of TPC2-A1-N (16) and analogs

2-Cyano-N-(4-(trifluoromethyl)phenyl)acetamide – SGA-34 (19)

According to general procedure A, 4-(trifluoromethyl)aniline (20, 812 µL, 6.47 mmol, 1.1 eq.), 2-cyanoacetic acid (18, 500 mg, 5.88 mmol, 1.0 eq.) and DCC (1.27 g, 6.17 mmol, 1.1 eq.) in DMF (7.0 mL) were used to yield amide SGA-34 (19) as colorless crystals (983 mg, 4.31 mmol, 73%). Analytical data are in accordance with literature[91, 179].

Rf = 0.14 (4:1 hexanes/acetone). m.p.: 195 °C. [lit.[91]: 191 – 193 °C]

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 10.65 (s, 1H, NH), 7.75 (d, J = 8.8 Hz, 2H, 3’-H, 5’-H), 7.70 (d, J = 8.8 Hz, 2H, 2’-H, 6’-H), 3.95 (s, 2H, 2-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 161.9 (C-1), 141.9 (C-1’), 126.3 (q, JCF = 3.7 Hz, C-

3’, C-5’), 124.4 (q, JCF = 271.4 Hz, CF3), 124.0 (q, JCF = 32.0 Hz, C-4’), 119.2 (C-2’, C-6’), 115.7 (CN), 27.0 (C-2).

-1 IR (ATR) ṽmax/cm = 3287, 3221, 3147, 1681, 1612, 1557, 1319, 1110, 1065, 849, 835.

- HRMS (ESI): calcd. for C10H6F3N2O (M-H) 227.04377; found 227.04371.

98 EXPERIMENTAL PART

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-N-(p-tolyl)acetamide (21)

According to general procedure A, p-toluidine (712 µL, 6.47 mmol, 1.1 eq.), 2-cyanoacetic acid (18, 500 mg, 5.88 mmol, 1.0 eq.) and DCC (1.27 g, 6.17 mmol, 1.1 eq.) in DMF (7.0 mL) were used to yield amide 21 as colorless crystals (728 mg, 4.18 mmol, 71%). Analytical data are in accordance with literature[180].

Rf = 0.14 (4:1 hexanes/acetone). m.p.: 184 °C. [lit.[180]: 186 °C]

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 10.19 (s, 1H, NH), 7.47 – 7.36 (m, 2H, 2’-H, 6’-H), 7.13

(d, J = 8.2 Hz, 2H, 3’-H, 5’-H), 3.86 (s, 2H, 2-H), 2.25 (s, 3H, CH3).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 160.7 (C-1), 135.9 (C-1’), 132.9 (C-4’), 129.3 (C-3’,

C-5’), 119.2 (C-2’, C-6’), 116.0 (CN), 26.6 (C-2), 20.4 (CH3).

-1 IR (ATR) ṽmax/cm = 3267, 3207, 3137, 1660, 1613, 1552, 1510, 819.

- HRMS (ESI): calcd. for C10H9N2O (M-H) 173.07204; found 173.07194.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-N-phenylacetamide (22)

According to general procedure A, aniline (1.96 mL, 21.5 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 2.01 g, 23.6 mmol, 1.1 eq.) and DCC (4.87 g, 23.6 mmol, 1.1 eq.) in DMF (20 mL) were used to yield amide 22 as colorless crystals (2.60 g, 16.2 mmol, 76%). Analytical data are in accordance with literature[180].

Rf = 0.12 (4:1 hexanes/acetone).

99 EXPERIMENTAL PART m.p.: 202 °C. [lit.[180]: 172 °C]

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 10.28 (s, 1H, NH), 7.54 (dt, J = 8.7, 1.6 Hz, 2H, 2’-H, 6’-H), 7.39 – 7.29 (m, 2H, 3’-H, 5’-H), 7.15 – 7.04 (m, 1H, 4’-H), 3.89 (s, 2H, 2-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 161.0 (C-1), 138.4 (C-1’), 128.9 (C-3’, C-5’), 123.9 (C- 4’), 119.2 (C-2’, C-6’), 115.9 (CN), 26.7 (C-2).

-1 IR (ATR) ṽmax/cm = 3265, 3207, 3143, 3099, 3052, 1653, 1620, 1557, 1299, 943, 761, 696.

- HRMS (ESI): calcd. for C9H7N2O (M-H) 159.05639; found 159.05628.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-N-(4-methoxyphenyl)acetamide (23)

According to general procedure A, p-anisidine (2.36 mL, 20.3 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 19.0 g, 22.3 mmol, 1.1 eq.) and DCC (4.61 g, 22.3 mmol, 1.1 eq.) in DMF (20 mL) were used to yield amide 23 as pale blue crystals (1.58 g, 8.31 mmol, 41%). Analytical data are in accordance with literature[180].

Rf = 0.10 (4:1 hexanes/acetone). m.p.: 137 °C. [lit.[180]: 176 °C]

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 10.14 (s, 1H, NH), 7.53 – 7.38 (m, 2H, 2’-H, 6’-H), 7.00

– 6.83 (m, 2H, 3’-H, 5’-H), 3.84 (s, 2H, 2-H), 3.72 (s, 3H, OCH3).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 160.4 (C-1), 155.6 (C-4’), 131.4 (C-1’), 120.8 (C-2’,

C-6’), 116.0 (CN), 114.0 (C-3’, C-5’), 55.2 (OCH3), 26.5 (C-2).

-1 IR (ATR) ṽmax/cm = 3299.3150, 1655, 1608, 1557, 1511, 1251, 1032, 828.

- HRMS (ESI): calcd. for C10H9N2O2 (M-H) 189.06695; found 189.06688.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

100 EXPERIMENTAL PART

N-(4-Chlorophenyl)-2-cyanoacetamide (24)

According to general procedure A, 4-chloroaniline (682 µL, 23.0 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 2.16 g, 25.4 mmol, 1.1 eq.) and DCC (5.23 g, 25.4 mmol, 1.1 eq.) in DMF (20 mL) were used to yield amide 24 as colorless crystals (3.46 g, 17.8 mmol, 77%). Analytical data are in accordance with literature[180].

Rf = 0.14 (3:2 hexanes/acetone). m.p.: 207 °C. [lit.[180]: 179 °C]

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 10.42 (s, 1H, NH), 7.62 – 7.51 (m, 2H, 2’-H, 6’-H), 7.50 – 7.27 (m, 2H, 3’-H, 5’-H), 3.91 (s, 2H, 2-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 161.2 (C-1), 137.3 (C-1’), 128.8 (C-3’, C-5’), 127.5 (C- 4’), 120.8 (C-2’, C-6’), 115.8 (CN), 26.8 (C-2).

-1 IR (ATR) ṽmax/cm = 3264, 3200, 3132, 3083, 1664, 1610, 1548, 1491, 832.

35 - HRMS (ESI): calcd. for C9H6 ClN2O (M-H) 193.01741; found 193.01750.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

N-(4-Bromophenyl)-2-cyanoacetamide (25)

According to general procedure A, 4-bromoaniline (2.00 mL, 17.1 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 1.60 g, 18.8 mmol, 1.1 eq.) and DCC (3.88 g, 18.8 mmol, 1.1 eq.) in DMF (20 mL) were used to yield amide 25 as colorless crystals (1.62 g, 6.76 mmol, 40%). Analytical data are in accordance with literature[180].

Rf = 0.14 (4:1 hexanes/acetone). m.p.: 186 °C. [lit.[180]: 185 °C]

101 EXPERIMENTAL PART

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 10.42 (s, 1H, NH), 7.52 (s, 4H, 2’-H, 3’-H, 5’-H, 6’-H), 3.90 (s, 2H, 2-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 161.2 (C-1), 137.7 (C-1‘), 131.7 (C-2‘, C-6’ or C-3’, C- 5’), 121.2 (C-2‘, C-6’ or C-3’, C-5’), 115.8 (C-4’), 115.5 (CN), 26.8 (C-2).

-1 IR (ATR) ṽmax/cm = 3322, 2927, 2849, 1608, 1547, 1245, 828.

79 - HRMS (ESI): calcd. for C9H6 BrN2O (M-H) 236.96690; found 236.96692.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-N-(4-fluorophenyl)acetamide (26)

According to general procedure A, 4-fluoroaniline (2.16 mL, 22.5 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 1.91 g, 22.5 mmol, 1.0 eq.) and DCC (4.64 g, 22.5 mmol, 1.0 eq.) in DMF (20 mL) were used to yield amide 26 as colorless crystals (3.00 g, 16.9 mmol, 75%). Analytical data are in accordance with literature[181].

Rf = 0.11 (4:1 hexanes/acetone). m.p.: 179 °C. [lit.[181]: 158 – 160 °C]

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 10.34 (s, 1H, NH), 7.66 – 7.46 (m, 2H, 2’-H, 6’-H), 7.27 – 7.09 (m, 2H, 3’-H, 5’-H), 3.89 (s, 2H, 2-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 161.0 (C-1), 158.3 (d, JCF = 240.5 Hz, C-4’), 134.7 (d,

JCF = 2.7 Hz, C-1’), 121.1 (d, JCF = 7.9 Hz, C-2’, C-6’), 115.9 (CN), 115.5 (d, JCF = 22.3 Hz, C- 3’, C-5’), 26.6 (C-2).

-1 IR (ATR) ṽmax/cm = 3274, 3166, 3107, 1662, 1623, 1566, 1505, 834.

- HRMS (ESI): calcd. for C9H6FN2O (M-H) 177.04696; found 177.04687.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

102 EXPERIMENTAL PART

2-Cyano-N-(4-iodophenyl)acetamide (27)

According to general procedure A, 4-iodoaniline (4.50 g, 20.5 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 17.5 g, 20.5 mmol, 1.0 eq.) and DCC (4.24 g, 20.5 mmol, 1.0 eq.) in DMF (20 mL) were used to yield amide 27 as pale blue crystals (4.50 g, 15.7 mmol, 77%). The compound is literature known, but no analytical data are available[91].

Rf = 0.11 (4:1 hexanes/acetone). m.p.: 218 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 10.38 (s, 1H, NH), 7.78 – 7.60 (m, 2H, 3’-H, 5’-H), 7.49 – 7.29 (m, 2H, 2’-H, 6’-H), 3.90 (s, 2H, 2-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 161.2 (C-1), 138.2 (C-1’), 137.6 (C-3’, C-5’), 121.4 (C- 2’, C-6’), 115.8 (CN), 87.6 (C-4’), 26.8 (C-2).

-1 IR (ATR) ṽmax/cm = 3265, 3188, 3113, 3078, 1666, 1543, 1391, 1299, 823.

- HRMS (ESI): calcd. for C9H6IN2O (M-H) 284.95303; found 284.95302.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-N-(4-nitrophenyl)acetamide (28)

According to general procedure A, 4-nitroaniline (696 µL, 7.24 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 616 mg, 7.24 mmol, 1.0 eq.) and DCC (1.49 g, 7.24 mmol, 1.0 eq.) in DMF (20 mL) were used to yield amide 28 as yellow solid (918 mg, 4.47 mmol, 62%). Analytical data are in accordance with literature[182].

Rf = 0.11 (4:1 hexanes/acetone). m.p.: 218 °C. [lit.[182]: 220 °C]

103 EXPERIMENTAL PART

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 10.88 (s, 1H, NH), 8.35 – 8.15 (m, 2H, 3’-H, 5’-H), 7.93 – 7.71 (m, 2H, 2’-H, 6’-H), 4.00 (s, 2H, 2-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 162.2 (C-1), 144.4 (C-1’), 142.7 (C-4’), 125.1 (C-3’, C-5’), 119.0 (C-2’, C-6’), 115.5 (CN), 27.2 (C-2).

-1 IR (ATR) ṽmax/cm = 3287, 1673, 1562, 1503, 1336, 1259, 860, 748.

- HRMS (ESI): calcd. for C9H6N3O3 (M-H) 204.04146; found 204.04146.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-N-(4-cyanophenyl)acetamide (29)

According to general procedure A, 4-aminobenzonitrile (1.00 g, 8.46 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 7.20 g, 8.46 mmol, 1.0 eq.) and DCC (1.75 g, 8.46 mmol, 1.0 eq.) in DMF (10 mL) were used to yield amide 29 as yellow solid (1.22 g, 6.57 mmol, 78%). The compound is literature known, but no analytical data are available[91].

Rf = 0.08 (4:1 hexanes/acetone). m.p.: 201 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 10.72 (s, 1H, NH), 7.82 – 7.78 (m, 2H, 3’-H, 5’-H), 7.74 – 7.70 (m, 2H, 2’-H, 6’-H), 3.97 (s, 2H, 2-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 162.0 (C-1), 142.5 (C-1’), 133.5 (C-3’, C-5’), 119.3 (C-

2’, C-6’), 118.9 (C-4’), 115.6 (CH2CN), 105.7 (CN), 27.1 (C-2).

-1 IR (ATR) ṽmax/cm = 3268, 3194, 3118, 2229, 1599, 1538, 1504, 845.

- HRMS (ESI): calcd. for C10H6N3O (M-H) 184.05164; found 184.05161.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

104 EXPERIMENTAL PART

N-(4-Acetylphenyl)-2-cyanoacetamide (30)

According to general procedure A, 4-aminoacetophenone (1.30 mL, 7.40 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 629 mg, 7.40 mmol, 1.0 eq.) and DCC (1.53 g, 7.40 mmol, 1.0 eq.) in DMF (10 mL) were used to yield amide 30 as yellow crystals (802 mg, 3.97 mmol, 54%). Analytical data are in accordance with literature[183].

Rf = 0.37 (3:2 hexanes/acetone). m.p.: 194 °C. [lit.[183]: 225 °C]

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 10.63 (s, 1H, NH), 7.99 – 7.91 (m, 2H, 3’-H, 5’-H), 7.74

– 7.62 (m, 2H, 2’-H, 6’-H), 3.96 (s, 2H, 2-H), 2.53 (s, 3H, CH3).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 196.5 (COCH3), 161.7 (C-1), 142.6 (C-1‘), 132.2 (C-

4‘), 129.6 (C-3‘, C-5‘), 118.5 (C-2‘, C-6‘), 115.7 (CN), 27.0 (C-2), 26.5 (CH3).

-1 IR (ATR) ṽmax/cm = 3286, 2250, 1695, 1651, 1599, 1536, 1279, 1249, 833, 720.

- HRMS (ESI): calcd. for C11H9N2O2 (M-H) 201.06695; found 201.06694.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-N-(4-propoxyphenyl)acetamide (31)

According to general procedure A, 4-propoxyaniline (679 µL, 4.49 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 382 mg, 4.49 mmol, 1.0 eq.) and DCC (927 mg, 4.49 mmol, 1.0 eq.) in DMF (2.0 mL) were used to yield amide 31 as colorless crystals (604 mg, 2.77 mmol, 62%).

Rf = 0.24 (95:5 CH2Cl2/EtOH). m.p.: 183 °C.

105 EXPERIMENTAL PART

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 10.13 (s, 1H, NH), 7.51 – 7.38 (m, 2H, 2’-H, 6’-H), 6.98

– 6.83 (m, 2H, 3’-H, 5’-H), 3.88 (t, J = 7.1 Hz, 2H, CH2CH2CH3), 3.84 (s, 2H, 2-H), 1.70 (sext,

J = 7.1 Hz, 2H, CH2CH2CH3), 0.96 (t, J = 7.1 Hz, 3H, CH2CH2CH3).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 160.4 (C-1), 155.1 (C-4’), 131.4 (C-1’), 120.8 (C-2’,

C-6’), 116.0 (CN), 114.5 (C-3’, C-5’), 69.0 (CH2CH2CH3), 26.5 (C-2), 22.0 (CH2CH2CH3), 10.4

(CH2CH2CH3).

-1 IR (ATR) ṽmax/cm = 3283, 3096, 1607, 1559, 1508, 1239, 828, 570.

- HRMS (ESI): calcd. for C12H13N2O2 (M-H) 217.09825; found 217.09832.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-N-(4-(trifluoromethoxy)phenyl)acetamide (32)

According to general procedure A, 4-(trifluoromethoxy)aniline (939 µL, 7.00 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 595 mg, 7.00 mmol, 1.0 eq.) and DCC (1.44 g, 7.00 mmol, 1.0 eq.) in DMF (10 mL) were used to yield amide 32 as colorless solid (1.23 g, 5.03 mmol, 72%). The compound is literature known, but no analytical data are available[91].

Rf = 0.39 (95:5 CH2Cl2/EtOH). m.p.: 154 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 10.49 (s, 1H, NH), 7.74 – 7.55 (m, 2H, 2’-H, 6’-H), 7.47 – 7.26 (m, 2H (3’-H, 5’-H), 3.92 (s, 2H, 2-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 161.3 (C-1), 144.0 (C-4‘), 137.5 (C-1‘), 121.8 (C-3’,

C-5’), 120.7 (C-2’, C-6’), 120.1 (q, JCF = 255.7 Hz, OCF3), 115.8 (CN), 26.8 (C-2).

-1 IR (ATR) ṽmax/cm = 3278, 2975, 1667, 1616, 1557, 1508, 1277, 1205, 1171.

- HRMS (ESI): calcd. for C10H6F3N2O2 (M-H) 243.03869; found 243.03869.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

106 EXPERIMENTAL PART

Methyl 4-(2-cyanoacetamido)benzoate (33)

According to general procedure A, methyl 4-aminobenzoate (3.00 g, 19.5 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 1.66 g, 19.5 mmol, 1.0 eq.) and DCC (4.01 g, 19.5 mmol, 1.0 eq.) in DMF (15 mL) were used to yield amide 33 as colorless solid (2.76 g, 12.6 mmol, 65%). The compound is literature known, but no analytical data are available[91].

Rf = 0.44 (3:2 hexanes/acetone). m.p.: 162 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 10.63 (s, 1H, NH), 8.01 – 7.87 (m, 2H, 2-H, 6-H), 7.76

– 7.61 (m, 2H, 3-H, 5-H), 3.96 (s, 2H, 2-H), 3.82 (s, 3H, CH3).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 165.7 (COOCH3), 161.7 (C-1’), 142.7 (C-4), 130.4 (C-

2, C-6), 124.6 (C-1), 118.6 (C-3, C-5), 115.7 (CN), 52.0 (CH3), 27.0 (C-2’).

-1 IR (ATR) ṽmax/cm = 2809, 1722, 1608, 1558, 1507, 1431, 1274, 1110, 757.

- HRMS (ESI): calcd. for C11H9N2O3 (M-H) 217.06187; found 217.06187.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

N-(2-Bromo-4-chlorophenyl)-2-cyanoacetamide (34)

According to general procedure A, 2-bromo-4-chloroaniline (1.00 g, 4.84 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 412 mg, 4.84 mmol, 1.0 eq.) and DCC (999 mg, 4.84 mmol, 1.0 eq.) in DMF (10 mL) were used to yield amide 34 as colorless crystals (919 mg, 3.36 mmol, 69%).

Rf = 0.22 (3:2 hexanes/acetone). m.p.: 157 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 9.97 (s, 1H, NH), 7.83 (d, J = 2.4 Hz, 1H, 3’-H), 7.63 (d, J = 8.7 Hz, 1H, 6’-H), 7.49 (dd, J = 8.7, 2.4 Hz, 1H, 5’-H), 3.98 (s, 2H, 2-H).

107 EXPERIMENTAL PART

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 161.8 (C-1), 134.7 (C-1’), 132.0 (C-3’), 130.7 (C-4’), 128.2 (C-5’), 128.0 (C-6’), 118.5 (C-2’), 115.7 (CN), 26.1 (C-2).

-1 IR (ATR) ṽmax/cm = 3281, 2258, 1666, 1577, 1530, 1470, 1284, 822.

79 35 - HRMS (ESI): calcd. for C9H5 Br ClN2O (M-H) 270.92793; found 270.92809.

Purity (HPLC): > 93% (λ = 210 nm), > 93% (λ = 254 nm).

2-Cyano-N-(2-iodophenyl)acetamide (35)

According to general procedure A, 2-iodoaniline (1.00 g, 4.57 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 388 mg, 4.57 mmol, 1.0 eq.) and DCC (942 mg, 4.57 mmol, 1.0 eq.) in DMF (10 mL) were used to yield amide 35 as brown crystals (913 g, 3.19 mmol, 70%).

Rf = 0.16 (4:1 hexanes/acetone). m.p.: 161 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 9.88 (s, 1H, NH), 7.90 (d, J = 7.6 Hz, 1H, 3’-H), 7.46 – 7.38 (m, 2H, 5’-H, 6’-H), 7.03 (ddd, J = 8.6, 5.4, 3.6 Hz, 1H, 4’-H), 3.93 (s, 2H, 2-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 161.4 (C-1), 139.1 (C-3’), 138.7 (C-1’), 128.8 (C-5’), 128.3 (C-4’), 127.4 (C-6’), 115.8 (CN), 96.4 (C-2’), 26.0 (C-2).

-1 IR (ATR) ṽmax/cm = 3252, 2263, 1658, 1577, 1542, 1433, 1015, 758, 768.

- HRMS (ESI): calcd. for C9H6IN2O (M-H) 284.95303; found 284.95295.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

N-(3-Chloro-2,4-difluorophenyl)-2-cyanoacetamide (36)

108 EXPERIMENTAL PART

According to general procedure A, 3-chloro-2,4-difluoroaniline (1.06 g, 6.48 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 551 mg, 6.48 mmol, 1.0 eq.) and DCC (1.34 g, 6.48 mmol, 1.0 eq.) in DMF (10 mL) were used to yield amide 36 as colorless crystals (1.01 g, 4.39 mmol, 68%).

Rf = 0.16 (4:1 hexanes/acetone). m.p.: 144 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 10.32 (s, 1H, NH), 7.79 (td, J = 9.0, 5.8 Hz, 1H, 6’-H), 7.33 (td, J = 9.0, 2.1 Hz, 1H, 5’-H), 3.99 (s, 2H, 2-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 162.0 (C-1), 154.8 (dd, JCF = 246.5, 2.0 Hz, C-2’ or C-

4’), 150.4 (dd, JCF = 250.4, 3.3 Hz, C-2’ or C-4’), 123.4 (dd, JCF = 8.8, 2.4 Hz, C-6’), 123.0 (dd,

JCF = 11.8, 3.5 Hz, C-1’), 115.7 (CN), 111.9 (dd, JCF = 21.4, 3.8 Hz, C-5’), 108.7 (dd, JCF = 21.9, 19.7 Hz, C-3’), 26.3 (C-2).

-1 IR (ATR) ṽmax/cm = 3274.2935, 2264, 1681, 1551, 1488, 1443, 1012, 831, 628.

35 - HRMS (ESI): calcd. for C9H4 ClF2N2O (M-H) 228.99857; found 228.99850.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-N-(3,4-dimethoxyphenyl)acetamide (37)

According to general procedure A, 3,4-dimethoxyaniline (1.00 g, 6.53 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 555 mg, 6.53 mmol, 1.0 eq.) and DCC (1.35 g, 6.53 mmol, 1.0 eq.) in DMF (10 mL) were used to yield amide 37 as violet crystals (1.10 g, 4.99 mmol, 76%). The compound is literature known, but no analytical data are available[184].

Rf = 0.05 (4:1 hexanes/acetone). m.p.: 174 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 10.15 (s, 1H, NH), 7.21 (d, J = 2.4 Hz, 1H, 2’-H), 7.04 (dd, J = 8.7, 2.4 Hz, 1H, 6’-H), 6.90 (d, J = 8.7 Hz, 1H, 5’-H), 3.84 (s, 2H, 2-H), 3.79 – 3.66 (m,

6H, 2x OCH3).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 160.5 (C-1), 148.6 (C-3’), 145.3 (C-4’), 131.9 (C-1’),

116.0 (CN), 112.0 (C-5’), 111.2 (C-6’), 104.3 (C-2’), 55.7 (OCH3), 55.4 (OCH3), 26.6 (C-2).

109 EXPERIMENTAL PART

-1 IR (ATR) ṽmax/cm = 3273, 2914, 2256, 1660, 1513, 1239, 1132, 1020, 837.

- HRMS (ESI): calcd. for C11H11N2O3 (M-H) 219.07752; found 219.07751.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-N-(2,3-dichlorophenyl)acetamide (38)

According to general procedure A, 2,3-dichloroaniline (745 µL, 6.30 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 536 mg, 6.30 mmol, 1.0 eq.) and DCC (1.30 g, 6.30 mmol, 1.0 eq.) in DMF (10 mL) were used to yield amide 38 as colorless crystals (414 mg, 1.81 mmol, 29%).

Rf = 0.21 (4:1 hexanes/acetone). m.p.: 176 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 10.11 (s, 1H, NH), 7.69 (dd, J = 8.1, 1.3 Hz, 1H, 4’-H or 6’-H), 7.51 (dd, J = 8.1, 1.3 Hz, 1H, 4’-H or 6’-H), 7.38 (t, J = 8.1 Hz, 1H, 5’-H), 4.02 (s, 2H, 2-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 161.9 (C-1), 136.1 (C-1’ or C-3’), 132.0 (C-1’ or C-3’), 128.2 (C-5’), 127.3 (C-4’ or C-6’), 125.3 (C-2’), 124.8 (C-4’ or C-6’), 115.7 (CN), 26.3 (C-2).

-1 IR (ATR) ṽmax/cm = 3287, 2253, 1666, 1580, 1527, 1415, 1338, 1182, 953, 788.

35 - HRMS (ESI): calcd. for C9H5 Cl2N2O (M-H) 226.97844; found 226.97859.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-N-(2,6-dibromophenyl)acetamide (39)

110 EXPERIMENTAL PART

According to general procedure A, 2,6-dibromoaniline (1.00 g, 4.00 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 340 mg, 4.00 mmol, 1.0 eq.) and DCC (825 mg, 4.00 mmol, 1.0 eq.) in DMF (10 mL) were used to yield amide 39 as colorless crystals (514 mg, 1.62 mmol, 40%).

Rf = 0.48 (3:2 hexanes/acetone). m.p.: 187 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 10.37 (s, 1H, NH), 7.74 (d, J = 8.1 Hz, 2H, 3’-H, 5’-H), 7.22 (t, J = 8.1 Hz, 1H, 4’-H), 3.93 (s, 2H, 2-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 161.1 (C-1), 134.7 (C-1’), 132.4 (C-3’, C-5’), 130.7 (C- 4’), 123.9 (C-2’, C-6’), 115.6 (CN), 25.4 (C-2).

-1 IR (ATR) ṽmax/cm = 3326, 2926, 2851, 1626, 1568, 1539, 1242, 642.

79 - HRMS (ESI): calcd. for C9H5 Br2N2O (M-H) 314.87741; found 314.87761.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

N-(3,5-Bis(trifluoromethyl)phenyl)-2-cyanoacetamide (40)

According to general procedure A, 3,5-bis(trifluoromethyl)aniline (1.09 mL, 7.00 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 595 mg, 7.00 mmol, 1.0 eq.) and DCC (1.44 g, 7.00 mmol, 1.0 eq.) in DMF (10 mL) were used to yield amide 40 as colorless crystals (1.68 g, 5.67 mmol, 81%). The compound is literature known, but no analytical data are available[185].

Rf = 0.43 (95:5 CH2Cl2/EtOH). m.p.: 141 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 10.96 (s, 1H, NH), 8.28 – 8.12 (m, 2H, 2’-H, 6’-H), 7.90 – 7.77 (m, 1H, 4’-H), 4.00 (s, 2H, 2-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 162.4 (C-1), 140.2 (C-1’), 130.9 (q, JCF = 32.9 Hz, C-

3’, C-5’), 123.1 (q, JCF = 272.7 Hz, CF3), 119.2 – 118.7 (m, C-2’, C-6’), 116.9 – 116.5 (m, C-4’), 115.4 (CN), 27.1 (C-2).

-1 IR (ATR) ṽmax/cm = 3313, 1695, 1572, 1471, 1381, 1272, 1132, 889, 703, 681.

111 EXPERIMENTAL PART

- HRMS (ESI): calcd. for C11H5F6N2O (M-H) 295.03116; found 295.03127.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-N-methyl-N-(4-(trifluoromethyl)phenyl)acetamide (41)

According to general procedure A, N-methyl-4-(trifluoromethyl)aniline (403 µL, 2.85 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 243 mg, 2.85 mmol, 1.0 eq.) and DCC (589 mg, 2.85 mmol, 1.0 eq.) in DMF (10 mL) were used to yield amide 41 as colorless solid (513 mg, 2.12 mmol, 74%). Analytical data are in accordance with literature[186].

Rf = 0.58 (95:5 CH2Cl2/EtOH). m.p.: 70 °C. [lit.[186]: 66-68 °C]

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 7.77 (d, J = 8.6 Hz, 2H, 3’-H, 5’-H), 7.40 (d, J = 8.2 Hz,

2H, 2’-H, 6’-H), 3.34 (s, 3H, CH3), 3.23 (s, 2H, 2-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 161.4 (C-1), 145.6 (C-1’), 131.7 (C-4’), 127.9 (C-2’,

C-6’ or C-3’, C-5’), 127.8 (C-2’, C-6’ or C-3’, C-5’), 123.5 (q, J = 271.4 Hz, CF3), 113.7 (CN),

38.1 (CH3), 25.6 (C-2).

-1 IR (ATR) ṽmax/cm = 3152, 2355, 1657, 1611, 1322, 1122, 1103, 1065, 848.

- HRMS (ESI): calcd. for C11H8F3N2O (M-H) 241.05942; found 241.05939.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-N-(4-(trifluoromethyl)benzyl)acetamide (42)

According to general procedure A, 4-(trifluoromethyl)benzylamine (2.50 mL, 17.5 mmol, 1.0 eq.), 2-cyanoacetic acid (18, 1.49 g, 17.5 mmol, 1.0 eq.) and DCC (3.62 g, 17.5 mmol, 1.0 eq.)

112 EXPERIMENTAL PART in DMF (15 mL) were used to yield amide 42 as colorless solid (299 mg, 1.23 mmol, 7%). Analytical data are in accordance with literature[187].

Rf = 0.46 (3:2 hexanes/acetone). m.p.: 113 °C. [lit.[187]: 128 °C]

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 8.83 (t, J = 5.7 Hz, 1H, NH), 7.74 – 7.66 (m, 2H, 3’-H,

5’-H), 7.54 – 7.44 (m, 2H, 2’-H, 6’-H), 4.38 (d, J = 5.7 Hz, 2H, CH2), 3.73 (s, 2H, 2-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 162.5 (C-1), 143.6 (C-1’), 128.0 (C-2’, C-6’), 127.7 (q,

JCF = 31.7 Hz, C-4’), 125.2 (q, JCF = 3.7 Hz, C-3’, C-5’). 124.3 (q, JCF = 272.1 Hz, CF3), 116.1

(CN), 42.2 (CH2), 25.3 (C-2).

-1 IR (ATR) ṽmax/cm = 3316, 2937, 2364, 1734, 1664, 1547, 1325, 1152, 1107, 1066.

- HRMS (ESI): calcd. for C11H8F3N2O (M-H) 241.05942; found 241.05949.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dichlorophenyl)-3-hydroxy-N-(4-(trifluoromethyl)phenyl)acrylamide – TPC2-A1-N (16)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to yield TPC2-A1-N (16) as colorless crystals (276 mg, 0.688 mmol, 69%).

Rf = 0.62 (4:1 hexanes/acetone). m.p.: 202 °C. [lit.[91]: 208 – 210 °C]

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 12.36 (s, 1H, NH), 7.79 – 7.76 (m, 2H, 2’-H, 6’-H), 7.65 (t, J = 1.9 Hz, 1H, 4’’-H), 7.62 – 7.60 (m, 2H, 3’-H, 5’-H), 7.59 (d, J = 1.9 Hz, 2H, 2’’-H, 6’’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 182.7 (C-3), 166.5 (C-1), 144.9 (C-1’’), 143.6 (C-1’),

133.5 (C-3’’, C-5’’), 128.6 (C-4’’), 126.2 – 125.9 (m, C-3’, C-5’ and C-2’’, C-6’’), 124.6 (q, JCF =

286.1 Hz, CF3), 123.3 (C-2), 121.8 (q, JCF = 31.8 Hz, C-4’), 118.7 (C-2’, C-6’), 77.7 (CN).

113 EXPERIMENTAL PART

-1 IR (ATR) ṽmax/cm = 3293, 2213, 1538, 1409, 1320, 1268, 1244, 1167, 1106, 1070, 837, 810, 660, 591.

35 - HRMS (ESI): calcd. for C17H8 Cl2F3N2O2 (M-H) 398.99204; found 398.99202.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dichlorophenyl)-3-hydroxy-N-phenylacrylamide – SGA-1 (44)

3' 4' 2' O OH 1' 2 2'' 1'' 3'' Cl 5' N 1 3 6' H CN 6'' 4'' 5'' 44 SGA-1 ( ) Cl

According to general procedure B, amide 22 (160 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-1 (44) as yellow solid (227 mg, 0.681 mmol, 68%).

Rf = 0.67 (3:2 hexanes/acetone). m.p.: 200 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 11.95 (s, 1H, NH), 7.61 (t, J = 1.9 Hz, 1H, 4’’-H), 7.56 (d, J = 1.9 Hz, 2H, 2’’-H, 6’’-H), 7.53 (dd, J = 8.5, 1.0 Hz, 2H, 2’-H, 6’-H), 7.27 – 7.22 (m, 2H, 3’-H, 5’-H), 6.96 – 6.90 (m, 1H, 4’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 182.2 (C-3), 166.1 (C-1), 145.3 (C-1’’), 140.1 (C-1’), 133.4 (C-3’’, C-5’’), 128.7 (C-3’, C-5’), 128.3 (C-4’’), 126.1 (C-2’’, C-6’’), 123.6 (C-2), 121.7 (C- 4’), 118.8 (C-2’, C-6’), 77.4 (CN).

-1 IR (ATR) ṽmax/cm = 3294, 2212, 1579, 1445, 810, 748, 683.

35 - HRMS (ESI): calcd. for C16H9 Cl2N2O2 (M-H) 331.00466; found 331.00465.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

N-(4-Chlorophenyl)-2-cyano-3-(3,5-dichlorophenyl)-3-hydroxyacrylamide – SGA-2 (45)

114 EXPERIMENTAL PART

According to general procedure B, amide 24 (195 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-2 (45) as yellow solid (256 mg, 0.695 mmol, 70%).

Rf = 0.71 (3:2 hexanes/acetone). m.p.: 230 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 12.12 (s, 2H, NH), 7.62 (t, J = 1.9 Hz, 1H, 4’’-H), 7.60 – 7.57 (m, 2H, 2’-H, 6’-H), 7.55 (d, J = 1.9 Hz, 2H, 2’’-H, 6’’-H), 7.30 – 7.26 (m, 2H, 3’-H, 5’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 182.5 (C-3), 166.1 (C-1), 145.4 (C-1’’), 139.1 (C-1’), 133.4 (C-3’’, C-5’’), 128.6 (C-3’, C-5’), 128.3 (C-4’’), 126.0 (C-2’’, C-6’’), 125.0 (C-4’), 123.6 (C- 2), 120.2 (C-2’, C-6’), 77.2 (CN).

-1 IR (ATR) ṽmax/cm = 3305, 2212, 1545, 1495, 1506, 1316, 1098, 809.

35 - HRMS (ESI): calcd. for C16H8 Cl3N2O2 (M-H) 364.96568; found 364.96593.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

N-(4-Bromophenyl)-2-cyano-3-(3,5-dichlorophenyl)-3-hydroxyacrylamide – SGA-3 (46)

According to general procedure B, amide 25 (239 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-3 (46) as yellow crystals (289 mg, 0.702 mmol, 70%).

Rf = 0.73 (3:2 hexanes/acetone). m.p.: 237 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.09 (s, 1H, NH), 7.62 (t, J = 1.9 Hz, 1H, 4’’-H), 7.58 – 7.51 (m, 4H, 2’-H, 6’-H, 2’’-H, 6’’-H), 7.44 – 7.38 (m, 2H, 3’-H, 5’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 182.4 (C-3), 166.1 (C-1), 145.2 (C-1’’), 139.4 (C-1’), 133.4 (C-3’’, C-5’’), 131.4 (C-3’, C-5’), 128.3 (C-4’’), 126.0 (C-2’’, C-6’’), 122.6 (C-2), 120.7 (C- 2’, C-6’), 112.9 (C-4’), 77.4 (CN).

-1 IR (ATR) ṽmax/cm = 3309, 2211, 1540, 1493, 1403, 1316, 1290, 1012, 809.

115 EXPERIMENTAL PART

79 35 - HRMS (ESI): calcd. for C16H8 Br Cl2N2O2 (M-H) 408.91517; found 408.91581.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dichlorophenyl)-3-hydroxy-N-(p-tolyl)acrylamide – SGA-4 (47)

According to general procedure B, amide 21 (174 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-4 (47) as yellow crystals (185 mg, 0.534 mmol, 53%).

Rf = 0.28 (3:2 hexanes/acetone). m.p.: 214 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 11.82 (s, 1H, NH), 7.63 (t, J = 1.9 Hz, 1H, 4’’-H), 7.56 (d, J = 1.9 Hz, 2H, 2’’-H, 6’’-H), 7.46 – 7.39 (m, 2H, 2’-H, 6’-H), 7.09 – 7.02 (m, 2H, 3’-H, 5’-H),

2.24 (s, 3H, CH3).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 182.0 (C-3), 166.0 (C-1), 145.1 (C-1’’), 137.4 (C-1’), 133.4 (C-3’’, C-5’’), 130.6 (C-4’), 129.1 (C-3’, C-5’), 128.3 (C-4’’), 126.1 (C-2’’, C-6’’), 123.5 (C-

2), 119.0 (C-2’, C-6’), 77.5 (CN), 20.4 (CH3).

-1 IR (ATR) ṽmax/cm = 3299, 2212, 1543, 1518.866, 806, 654.

35 - HRMS (ESI): calcd. for C17H11 Cl2N2O2 (M-H) 345.02031; found 345.02052.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dichlorophenyl)-N-(4-fluorophenyl)-3-hydroxyacrylamide – SGA-8 (48)

116 EXPERIMENTAL PART

According to general procedure B, amide 26 (178 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-8 (48) as white crystals (220 mg, 0.626 mmol, 63%).

Rf = 0.69 (3:2 hexanes/acetone). m.p.: 225 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 11.92 (s, 1H, NH), 7.63 (t, J = 1.9 Hz, 1H, 4’’-H), 7.60 – 7.53 (m, 4H, 2’-H, 6’-H, 2’’-H, 6’’-H), 7.12 – 7.03 (m, 2H, 3’-H, 5’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 182.2 (C-3), 166.1 (C-1), 157.3 (d, JCF = 236.9 Hz, C-

4’), 144.9 (C-1’’), 136.3 (d, JCF = 2.4 Hz, C-1’), 133.4 (C-3’’, C-5’’), 128.4 (C-4’’), 126.1 (C-2’’,

C-6’’), 123.3 (C-2), 120.5 (d, JCF = 7.6 Hz, C-2’, C-6’), 115.2 (d, JCF = 22.0 Hz, C-3’, C-5’), 77.4 (CN).

-1 IR (ATR) ṽmax/cm = 3296, 2212, 1739, 1549, 1506, 1210, 823, 809, 645.

35 - HRMS (ESI): calcd. for C16H8 Cl2FN2O2 (M-H) 348.99523; found 348.99526.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dichlorophenyl)-N-(4-iodophenyl)-3-hydroxyacrylamide – SGA-9 (49)

According to general procedure B, amide 27 (286 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-9 (49) as yellow crystals (279 mg, 0.608 mmol, 61%).

Rf = 0.71 (3:2 hexanes/acetone). m.p.: 238 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.04 (s, 1H, NH), 7.63 (t, J = 1.9 Hz, 1H, 4’’-H), 7.59 – 7.53 (m, 4H, 3’-H, 5’-H, 2’’-H, 6’’-H), 7.44 – 7.38 (m, 2H, 2’-H, 6’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 182.4 (C-3), 166.2 (C-1), 145.1 (C-1’’), 139.8 (C-1’), 137.3 (C-3’, C-5’ or C-2’’, C-6’’), 133.4 (C-3’’, C-5’’), 128.4 (C-4’’), 126.1 (C-3’, C-5’ or C-2’’, C- 6’’), 123.3 (C-2), 121.1 (C-2’, C-6’), 84.5 (C-4’), 77.5 (CN).

117 EXPERIMENTAL PART

-1 IR (ATR) ṽmax/cm = 3303, 2217, 1592, 1523, 1485, 1314, 817, 806, 658.

35 - HRMS (ESI): calcd. for C16H8 Cl2IN2O2 (M-H) 456.90130; found 456.90014.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-hydroxy-3-phenyl-N-(4-(trifluoromethyl)phenyl)acrylamide – SGA-10 (50)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and benzoyl chloride (128 µL, 1.10 mmol, 1.1 eq.) were used to give SGA-10 (50) as colorless crystals (185 mg, 0.557 mmol, 56%). Analytical data are in accordance with literature[179].

Rf = 0.28 (3:2 hexanes/acetone). m.p.: 242 °C. [lit.[179]: 245 – 247 °C]

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.12 (s, 1H, NH), 7.84 – 7.74 (m, 2H, 2’-H, 6’-H), 7.70 – 7.58 (m, 4H, 3’-H, 5’-H, Ph), 7.48 – 7.39 (m, 3H, Ph).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 185.7 (C-3), 167.3 (C-1), 143.1 (C-1’), 139.7 (qPh),

135.0 (C-2), 130.0 (Ph), 127.8 (Ph), 127.6 (Ph), 126.0 (q, JCF = 3.8 Hz, C-3’, C-5’), 125.0 (d,

JCF = 270.6 Hz, CF3), 122.3 (d, JCF = 35.2 Hz, C-4’), 119.4 (C-2’, C-6’), 77.8 (CN).

-1 IR (ATR) ṽmax/cm = 3283, 2216, 1592, 1550, 1309, 1109, 1067, 840, 694.

- HRMS (ESI): calcd. for C17H10F3N2O2 (M-H) 331.06999; found 331.06985.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dinitrophenyl)-3-hydroxy-N-(4-(trifluoromethyl)phenyl)acrylamide – SGA-11 (51)

3' F C 4' 3 2' O OH 1' 2 2'' 1'' 3'' NO2 5' N 1 3 6' H CN 6'' 4'' 5'' SGA-11 (51) NO2

118 EXPERIMENTAL PART

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dinitrobenzoyl chloride (254 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-11 (51) as red crystals (124 mg, 0.294 mmol, 29%).

Rf = 0.21 (3:2 hexanes/acetone). m.p.: 240 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 12.32 (s, 1H, NH), 10.00 (s, 1H, OH), 8.89 – 8.80 (m, 3H, 2’’-H, 4’’-H, 6’’-H), 7.84 – 7.75 (m, 2H, 2’-H, 6’-H), 7.67 – 7.58 (m, 2H, 3’-H, 5’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 180.5 (C-3), 166.1 (C-1), 147.6 (C-3’’, C-5’’), 144.4

(C-1’’), 143.5 (C-1’), 127.6 (C-2’’, C-6’’),126.1 (q, JCF = 3.5 Hz, C-3’, C-5’),124.2 (q, JCF = 271.0

Hz, CF3), 123.3 (C-2), 121.8 (q, JCF = 31.7 Hz, C-4’), 119.0 (C-4’’), 118.6 (C-2’, C-6’), 77.7 (CN).

-1 IR (ATR) ṽmax/cm = 3262, 3093, 2223, 1539, 1342, 1317, 1115, 1067, 841, 730, 703, 687.

- HRMS (ESI): calcd. for C17H8F3N4O6 (M-H) 421.04014; found 421.04021.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dichlorophenyl)-N-(4-methoxyphenyl)-3-hydroxyacrylamide – SGA-12 (52)

According to general procedure B, amide 23 (190 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-12 (52) as yellow solid (220 mg, 0.606 mmol, 61%).

Rf = 0.76 (3:2 hexanes/acetone). m.p.: 207 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 11.36 (s, 1H, NH), 9.87 (s, 1H, OH), 7.71 (t, J = 1.8 Hz, 1H, 4’-H), 7.65 (d, J = 1.8 Hz, 2H, 2’’-H, 6’’-H), 7.49 – 7.41 (m, 2H, 2’-H, 6’-H), 6.90 – 6.85

(m, 2H, 3’-H, 5’-H), 3.72 (s, 3H, OCH3).

119 EXPERIMENTAL PART

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 181.6 (C-3), 166.4 (C-1), 155.2 (C-4’), 142.6 (C-1’’), 133.7 (C-3’’, C-5’’), 131.9 (C-1’), 129.3 (C-4’’), 126.3 (C-2’’, C-6’’), 121.7 (C-2’, C-6’), 121.4 (C-

2), 113.9 (C-3’, C-5’), 78.1 (CN), 55.2 (OCH3).

-1 IR (ATR) ṽmax/cm = 3296, 2211, 1601, 1467, 1441, 1297, 1251, 1032, 764.

35 - HRMS (ESI): calcd. for C17H11 Cl2N2O3 (M-H) 361.01522; found 361.01516.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dichlorophenyl)-N-(4-nitrophenyl)-3-hydroxyacrylamide – SGA-13 (53)

According to general procedure B, amide 28 (205 mg, 1.00 mmol, 1.0 eq.) in dry THF (16 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-13 (53) as yellow solid (208 mg, 0.550 mmol, 55%).

Rf = 0.82 (3:2 hexanes/acetone). m.p.: 246 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.64 (s, 1H, NH), 8.19 – 8.13 (m, 2H, 3’-H, 5’-H), 7.82 – 7.77 (m, 2H, 2’-H, 6’-H), 7.64 (t, J = 1.9 Hz, 1H, 4’’-H), 7.57 (d, J = 1.9 Hz, 2H, 2’’-H, 6’’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 181.6 (C-3), 166.9 (C-1), 149.4 (C-1’’), 147.0 (C-1’), 141.3 (C-4’), 133.9 (C-3’’, C-5’’), 129.0 (C-4’’), 126.5 (C-2’’, C-6’’), 125.7 (C-3’, C-5’), 121.8 (C- 2), 118.6 (C-2’, C-6’), 77.0 (CN).

-1 IR (ATR) ṽmax/cm = 3314, 2209, 1568, 1546, 1514, 1498, 1340, 1309, 847, 813, 656.

35 - HRMS (ESI): calcd. for C16H8 Cl2N3O4 (M-H) 375.98973; found 375.98970.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

120 EXPERIMENTAL PART

2-Cyano-3-(4-nitrophenyl)-3-hydroxy-N-(4-(trifluoromethyl)phenyl)acrylamide – SGA-15 (54)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 4-nitrobenzoyl chloride (204 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-15 (54) as yellow crystals (276 mg, 0.688 mmol, 69%).

Rf = 0.19 (3:2 hexanes/acetone). m.p.: 217 °C. [lit.[91]: 211 – 214 °C]

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 12.39 (s, 1H, NH), 8.28 – 8.19 (m, 2H, 3’’-H, 5’’-H), 7.84 – 7.80 (m, 2H, 2’’-H, 6’’-H), 7.78 (d, J = 8.6 Hz, 2H, 2’-H, 6’-H), 7.60 (d, J = 8.6 Hz, 2H, 3’’-H, 5’’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 184.3 (C-3), 166.4 (C-1), 148.1 (C-1’’), 147.5 (C-4’’),

143.7 (C-1’), 128.6 (C-2’’, C-6’’), 126.1 (q, JCF = 3.6 Hz, C-3’, C-5’), 124.6 (q, JCF = 270.9 Hz,

CF3), 123.2 (C-2), 123.1 (C-3’’, C-5’’), 121.6 (q, JCF = 32.0 Hz, C-4’), 118.6 (C-2’, C-6’), 77.9 (CN).

-1 IR (ATR) ṽmax/cm = 3307, 2219, 1551, 1320, 1111, 1069, 844, 750, 700.

- HRMS (ESI): calcd. for C17H9F3N3O4 (M-H) 376.05506; found 376.05509.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

3-(4-Chlorophenyl)-2-cyano-3-hydroxy-N-(4-(trifluoromethyl)phenyl)acrylamide – SGA-16 (55)

3' F C 4' 3 2' O OH 1' 2 2'' 1'' 3'' 5' N 1 3 6' H CN 6'' 4'' Cl 55 5'' SGA-16 ( )

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 4-chlorobenzoyl chloride (141 µL, 1.10 mmol, 1.1 eq.) were used to give SGA-16 (55) as colorless crystals (222 mg, 0.605 mmol, 61%).

121 EXPERIMENTAL PART

Rf = 0.32 (3:2 hexanes/acetone). m.p.: 220 °C. [lit.[91]: 218 – 220 °C]

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 12.31 (s, 1H, NH), 7.81 – 7.75 (m, 2H, 2’-H, 6’-H), 7.68 – 7.63 (m, 2H, 2’’-H, 6’’-H), 7.63 – 7.58 (m, 2H, 3’-H, 5’-H), 7.49 – 7.44 (m, 2H, 3’’-H, 5’’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 184.8 (C-3), 167.0 (C-1), 143.5 (C-1’), 139.6 (C-1’’),

134.1 (C-4’’), 129.4 (C-2’’, C-6’’), 127.8 (C-3’’, C-5’’), 126.0 (q, JCF = 3.6 Hz, C-3’, C-5’), 124.6

(q, JCF = 271.0 Hz, CF3), 123.0 (C-2), 121.8 (q, JCF = 31.0 Hz, C-4’), 118.9 (C-2’, C-6’), 77.5 (CN).

-1 IR (ATR) ṽmax/cm = 3282.2215, 1587, 1240, 1302, 1129, 1113, 1097, 839.

35 - HRMS (ESI): calcd. for C17H9 ClF3N2O2 (M-H) 365.03101; found 365.03108.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

N-(2-Bromo-4-chlorophenyl)-2-cyano-3-(3,5-dichlorophenyl)-3-hydroxyacrylamide – SGA-27 (56)

According to general procedure B, amide 34 (274 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-27 (56) as colorless solid (330 mg, 0.738 mmol, 74%).

Rf = 0.25 (3:2 hexanes/acetone). m.p.: 203 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 12.29 (s, 1H, NH), 8.56 (d, J = 9.0 Hz, 1H, 6’-H), 7.68 (d, J = 2.5 Hz, 1H, 3’-H), 7.63 (t, J = 1.9 Hz, 1H, 4’’-H), 7.58 (d, J = 1.9 Hz, 2H, 2’’-H, 6’’-H), 7.36 (dd, J = 9.0, 2.5 Hz, 1H, 5’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 182.7 (C-3), 166.4 (C-1), 145.2 (C-1’’), 137.8 (C-1’), 133.4 (C-3’’, C-5’’), 131.4 (C-6’), 128.4 (C-4’’), 127.8 (C-5’), 126.1 (C-2’’, C-6’’), 125.4 (C-4’), 123.5 (C-2), 122.1 (C-3’), 112.1 (C-2’), 77.3 (CN).

-1 IR (ATR) ṽmax/cm = 3366, 3087, 2208, 1556, 1521, 1469, 1360, 1292, 863, 815.

122 EXPERIMENTAL PART

79 35 - HRMS (ESI): calcd. for C16H7 Br Cl3N2O2 (M-H) 442.87620; found 442.87736.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dichlorophenyl)-N-(3,4-dimethoxyphenyl)-3-hydroxyacrylamide – SGA-28 (57)

According to general procedure B, amide 37 (220 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-28 (57) as yellow solid (200 mg, 0.508 mmol, 51%).

Rf = 0.17 (3:2 hexanes/acetone). m.p.: 195 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 11.52 (s, 1H, NH), 7.68 (t, J = 1.8 Hz, 1H, 4’’-H), 7.62 (d, J = 1.8 Hz, 2H, 2’’-H, 6’’-H), 7.30 (d, J = 2.2 Hz, 1H, 2’-H), 7.01 (dd, J = 8.7, 2.2 Hz, 1H, 6’-

H), 6.86 (d, J = 8.7 Hz, 1H, 5’-H), 3.73 (s, 3H, OCH3), 3.71 (s, 3H, OCH3).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 181.7 (C-3), 166.2 (C-1), 148.6 (C-3’), 144.4 (C-4’), 143.6 (C-1’’), 133.6 (C-3’’, C-5’’), 133.0 (C-1’), 128.9 (C-4’’), 126.2 (C-2’’, C-6’’), 122.2 (C-2),

112.3 (C-5’), 111.5 (C-6’), 104.9 (C-2’), 77.9 (CN), 55.8 (OCH3), 55.4 (OCH3).

-1 IR (ATR) ṽmax/cm = 3284, 2217, 1608, 1549, 1516, 1238, 1028, 810.

35 - HRMS (ESI): calcd. for C18H13 Cl2N2O4 (M-H) 391.02579; found 391.02608.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(1-methyl-1H-pyrrol-2-yl)-3-hydroxy-N-phenylacrylamide (prinomide) – SGA-31 (58)

123 EXPERIMENTAL PART

According to general procedure B, amide 22 (228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 1-methylpyrrole-2-carbonyl chloride (158 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-31 (58) as colorless crystals (200 mg, 0.747 mmol, 75%).

Rf = 0.65 (3:2 hexanes/acetone). m.p.: 171 °C. [lit.[188]: 174 – 175 °C]

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.74 (s, 1H, NH), 7.54 (dd, J = 4.3, 1.6 Hz, 1H, 3’’-H), 7.50 (d, J = 8.0 Hz, 2H, 2’-H, 6’-H), 7.44 – 7.34 (m, 2H, 3’-H, 5’-H), 7.20 (t, J = 7.4 Hz, 1H, 4’-H),

6.89 (t, J = 1.9 Hz, 1H, 5’’-H), 6.25 (dd, J = 4.3, 2.5 Hz, 1H, 4’’-H), 3.92 (s, 3H, CH3).

13 C NMR (101 MHz, CDCl3) δ/ppm = 175.6 (C-3), 170.0 (C-1), 136.2 (C-1’), 132.0 (C-5’’), 129.3 (C-3’, C-5’), 125.7 (C-4’), 124.4 (C-2’’), 121.3 (C-2’, C-6’), 120.7 (C-3’’), 119.1 (C-2), 109.7 (C-

4’’), 72.6 (CN), 38.5 (CH3).

-1 IR (ATR) ṽmax/cm = 3293, 2210, 1526, 1382, 1231, 751, 736, 687.

- HRMS (ESI): calcd. for C15H12N3O2 (M-H) 266.09350; found 266.09370.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(1-methyl-1H-pyrrol-2-yl)-3-hydroxy-N-(4-(trifluoromethyl)phenyl)acrylamide – SGA-32 (59)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 1-methylpyrrole-2-carbonyl chloride (158 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-32 (59) as colorless crystals (221 mg, 0.660 mmol, 66%).

Rf = 0.57 (3:2 hexanes/acetone). m.p.: 203 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.86 (s, 1H, NH), 7.69 – 7.61 (m, 4H, 2’-H, 6’-H, 3’-H, 5’- H), 7.57 (dd, J = 4.3, 1.6 Hz, 1H, 3’’-H), 6.93 – 6.90 (m, 1H, 4’’-H), 6.27 (dd, J = 4.3, 2.5 Hz,

1H, 5’’-H), 3.93 (s, 3H, CH3).

124 EXPERIMENTAL PART

13 C NMR (101 MHz, CDCl3) δ/ppm = 175.6 (C-3), 170.2 (C-1), 139.5 (C-1’), 132.6 (C-5’’), 127.4

(q, JCF = 31.8 Hz, C-4’), 126.6 (q, J = 3.8 Hz, C-3’, C-5’), 124.1 (C-2’’), 124.0 (q, JCF = 271.4

Hz, CF3), 121.3 (C-3’’), 120.7 (C-2’, C-6’), 119.0 (C-2), 110.0 (C-4’’), 72.6 (CN), 38.7 (CH3).

-1 IR (ATR) ṽmax/cm = 3273, 2210, 1518, 1379, 1228, 1111, 996, 837, 745.

- HRMS (ESI): calcd. for C16H11F3N3O2 (M-H) 334.08088; found 334.08090.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

N-(3-Chloro-2,4-difluorophenyl)-2-cyano-3-(3,5-dichlorophenyl)-3-hydroxyacrylamide – SGA-33 (60)

According to general procedure B, amide 36 (231 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-33 (60) as colorless crystals (290 mg, 0.719 mmol, 72%).

Rf = 0.15 (3:2 hexanes/acetone). m.p.: 193 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.34 (s, 1H, NH), 8.43 (td, J = 9.0, 6.0 Hz, 1H, 6’-H), 7.63 (t, J = 1.9 Hz, 1H, 4’’-H), 7.58 (d, J = 1.9 Hz, 2H, 2’’-H, 6’’-H), 7.23 (td, J = 9.2, 2.1 Hz, 1H, 5’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 182.9 (C-3), 166.3 (C-1), 152.1 (d, JCF = 242.4 Hz, C-

4’), 147.7 (d, JCF = 245.4 Hz, C-2’), 145.0 (C-1’’), 133.4 (C-3’’, C-5’’), 128.5 (C-4’’), 126.3 (dd,

JCF = 10.3, 2.6 Hz, C-1’), 126.1 (C-2’’, C-6’’), 123.2 (C-2), 119.1 (dd, JCF = 7.8, 3.0 Hz, C-6’),

111.5 (dd, JCF = 20.7, 3.6 Hz, C-5’), 107.8 (dd, JCF = 22.2, 2.9 Hz, C-3’), 77.3 (CN).

-1 IR (ATR) ṽmax/cm = 3293, 2206, 1539, 1497, 1370, 1289, 1023, 803.

35 - HRMS (ESI): calcd. for C16H6 Cl3F2N2O2 (M-H) 400.94684; found 400.94724.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

125 EXPERIMENTAL PART

2-Cyano-N-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-3-hydroxyacrylamide – SGA-38 (61)

According to general procedure B, amide 29 (185 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-38 (61) as yellow solid (137 mg, 0.382 mmol, 38%).

Rf = 0.12 (3:2 hexanes/acetone). m.p.: 236 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 12.42 (s, 1H, NH), 7.75 – 7.72 (m, 2H, 3’-H, 5’-H), 7.71 – 7.67 (m, 2H, 2’-H, 6’-H), 7.64 (t, J = 1.9 Hz, 1H, 4’’-H), 7.57 (d, J = 1.9 Hz, 2H, 2’’-H, 6’’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 183.0 (C-3), 166.4 (C-1), 145.1 (C-1’’), 144.3 (C-1’), 133.5 (C-3’’, C-5’’), 133.3 (C-3’, C-5’), 128.5 (C-4’’), 126.0 (C-2’’, C-6’’), 123.2 (C-4’), 119.4 (C- 2), 118.8 (C-2’, C-6’), 103.1 (4’-CN), 77.4 (2-CN).

-1 IR (ATR) ṽmax/cm = 3321, 2360, 2340, 1533, 839, 655.

35 - HRMS (ESI): calcd. for C17H8 Cl2N3O2 (M-H) 355.99991; found 356.00057.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dichlorophenyl)-3-hydroxy-N-(2-iodophenyl)acrylamide – SGA-39 (62)

According to general procedure B, amide 35 (286 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-39 (62) as yellow solid (237 mg, 0.515 mmol, 52%).

Rf = 0.20 (3:2 hexanes/acetone). m.p.: 171 °C.

126 EXPERIMENTAL PART

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 11.87 (s, 1H, NH), 8.27 (dd, J = 8.3, 1.5 Hz, 1H, 6’-H), 7.80 (dd, J = 7.9, 1.5 Hz, 1H, 3’-H), 7.63 (t, J = 1.9 Hz, 1H, 4’’-H), 7.59 (d, J = 1.9 Hz, 2H, 2’’- H, 6’’-H), 7.29 (ddd, J = 8.4, 7.3, 1.5 Hz, 1H, 5’-H), 6.80 – 6.67 (m, 1H, 4’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 182.2 (C-3), 166.4 (C-1), 145.1 (C-1’’), 141.5 (C-1’), 139.0 (C-3’), 133.4 (C-3’’, C-5’’), 128.4 (C-4’’), 128.3 (C-5’), 126.1 (C-2’’, C-6’’), 124.0 (C-4’), 123.6 (C-2), 122.1 (C-6’), 89.2 (C-2’), 77.2 (CN).

-1 IR (ATR) ṽmax/cm = 3337, 2213, 1579, 1537, 1294, 742.

35 - HRMS (ESI): calcd. for C16H8 Cl2IN2O2 (M-H) 456.90130; found 456.90095.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-hydroxy-3-(perfluorophenyl)-N-(4-(trifluoromethyl)phenyl)acrylamide – SGA-40 (63)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 2,3,4,5,6-pentafluorobenzoyl chloride (158 µL, 1.10 mmol, 1.1 eq.) were used to give SGA-40 (63) as colorless crystals (236 mg, 0.560 mmol, 56%).

Rf = 0.28 (3:2 hexanes/acetone). m.p.: 161 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 11.70 (s, 1H, NH), 7.81 – 7.72 (m, 2H, 2’-H, 6’-H), 7.67 – 7.56 (m, 2H, 3’-H, 5’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 172.6 (C-3), 164.9 (C-1), 143.9 – 140.9 (m, C-2’’, C- 6’’ or C-3’’, C-5’’), 143.3 (C-1’), 141.9 – 138.9 (m, C-4’’), 138.5 – 135.1 (m, C-2’’, C-6’’ or C-3’’,

C-5’’), 126.1 (q, JCF = 3.6 Hz, C-3’, C-5’), 124.6 (q, JCF = 271.0 Hz, CF3), 122.0 (q, JCF = 31.9 Hz, C-4’), 117.5 – 117.0 (m, C-1’’), 121.7 (C-2’, C-6’), 118.7 (C-2), 81.8 (CN).

-1 IR (ATR) ṽmax/cm = 2230, 1590, 1543, 1524, 1497, 1323, 1116, 1000, 839.

- HRMS (ESI): calcd. for C17H5F8N2O2 (M-H) 421.02288; found 421.02337.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

127 EXPERIMENTAL PART

2-Cyano-3-(3,5-dibromophenyl)-3-hydroxy-N-(4-(trifluoromethyl)phenyl)acrylamide – SGA-70 (64)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dibromobenzoic acid (308 mg, 1.10 mmol, 1.1 eq., converted into the corresponding aryl chloride) were used to give SGA-70 (64) as light yellow crystals (206 mg, 0.420 mmol, 42%).

Rf = 0.32 (3:2 hexanes/acetone). m.p.: 211 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.32 (s, 1H, NH), 8.96 (s, 1H, OH), 7.91 – 7.81 (m, 1H, 4’’-H), 7.78 – 7.72 (m, 4H, 2’-H, 6’-H, 2’’-H, 6’’-H), 7.65 – 7.55 (m, 2H, 3’-H, 5’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 182.5 (C-3), 166.4 (C-1), 145.5 (C-1’’), 143.6 (C-1’),

133.7 (C-4’’), 129.2 (C-2’’, C-6’’), 126.0 (q, JCF = 3.5 Hz, C-3’, C-5’), 123.9 (q, JCF = 271.2 Hz,

CF3), 123.3 (C-2), 121.8 (C-3’’, C-5’’), 121.6 (q, JCF = 31.9 Hz, C-4’), 118.5 (C-2’, C-6’), 77.4 (CN).

-1 IR (ATR) ṽmax/cm = 2218, 1594, 1538, 1315, 1166, 1109, 1068, 838, 750.

79 - HRMS (ESI): calcd. for C17H8 Br2F3N2O2 (M-H) 486.89101; found 486.89128.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-hydroxy-3-(2,4,6-trichlorophenyl)-N-(4-(trifluoromethyl)phenyl)acrylamide – SGA-71 (65)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 2,4,6-trichlorobenzoyl chloride (172 µL,

128 EXPERIMENTAL PART

1.10 mmol, 1.1 eq.) were used to give SGA-71 (65) as colorless crystals (158 mg, 0.363 mmol, 36%).

Rf = 0.14 (3:2 hexanes/acetone). m.p.: 220 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 11.92 (s, 1H, NH), 7.79 – 7.74 (m, 2H, 2’-H, 6’-H), 7.66 (s, 2H, 3’’-H, 5’’-H), 7.63 – 7.58 (m, 2H, 3’-H, 5’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 180.7 (C-3), 165.7 (C-1), 143.6 (C-1’), 139.5 (C-1’’),

133.1 (C-4’’), 132.0 (C-2’’, C-6’’), 127.8 (C-3’’, C-5’’), 126.1 (q, JCF = 3.6 Hz, C-3’, C-5’), 124.6

(q, JCF = 271.1 Hz, CF3), 121.9 (C-2), 121.6 (q, JCF = 31.9 Hz, C-4’), 118.4 (C-2’, C-6’), 79.5 (CN).

-1 IR (ATR) ṽmax/cm = 2230, 1598, 1541, 1318, 1116, 841.

35 - HRMS (ESI): calcd. for C17H7 Cl3F3N2O2 (M-H) 432.95307; found 432.95394.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

N-(4-Bromophenyl)-2-cyano-3-hydroxy-3-(2,4,6-trichlorophenyl)acrylamide – SGA-72 (66)

According to general procedure B, amide 25 (239 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 2,4,6-trichlorobenzoyl chloride (172 µL, 1.10 mmol, 1.1 eq.) were used to give SGA-72 (66) as colorless solid (227 mg, 0.507 mmol, 51%).

Rf = 0.14 (3:2 hexanes/acetone). m.p.: 191 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 11.69 (s, 1H, NH), 7.65 (s, 2H, 3’’-H, 5’’-H), 7.58 – 7.52 (m, 2H, 2’-H, 6’-H), 7.44 – 7.38 (m, 2H, 3’-H, 5’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 180.3 (C-3), 165.4 (C-1), 139.6 (C-1’ or C-1’’), 139.4 (C-1’ or C-1’’), 133.0 (C-4’’), 132.0 (C-2’’, C-6’’), 131.5 (C-3’, C-5’), 127.8 (C-3’’, C-5’’), 122.1 (C-2), 120.5 (C-2’, C-6’), 112.9 (C-4’), 79.5 (CN).

129 EXPERIMENTAL PART

-1 IR (ATR) ṽmax/cm = 2238, 1588, 1539, 1488, 1307, 856, 818.

79 35 - HRMS (ESI): calcd. for C16H7 Br Cl3N2O2 (M-H) 442.87620; found 442.87747.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

N-(3-Chloro-2,4-difluorophenyl)-2-cyano-3-hydroxy-3-(2,4,6-trichlorophenyl)acrylamide – SGA-73 (67)

According to general procedure B, amide 36 (231 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 2,4,6-trichlorobenzoyl chloride (172 µL, 1.10 mmol, 1.1 eq.) were used to give SGA-73 (67) as colorless solid (217 mg, 0.496 mmol, 50%).

Rf = 0.16 (1:1 hexanes/acetone). m.p.: 199 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 11.93 (s, 1H, NH), 8.45 (td, J = 9.1, 5.9 Hz, 1H, 6’-H), 7.66 (s, 2H, 3’’-H, 5’’-H), 7.23 (td, J = 9.2, 2.0 Hz, 1H, 5’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 180.8 (C-3), 165.6 (C-1), 152.1 (d, JCF = 242.6 Hz, C-

4’), 147.5 (d, JCF = 245.6 Hz, C-2’), 139.4 (C-1’’ or C-4’’), 133.1 (C-1’’ or C-4’’), 132.0 (C-2’’, C-

6’’), 127.8 (C-3’’, C-5’’), 126.3 (dd, JCF = 10.2, 3.2 Hz, C-1’), 121.8 (C-2), 118.8 (dd, JCF = 7.9,

2.9 Hz, C-6’), 111.5 (dd, JCF = 20.7, 3.5 Hz, C-5’), 107.9 (dd, JCF = 22.0, 19.2 Hz, C-3’), 79.4 (CN).

-1 IR (ATR) ṽmax/cm = 3279, 2222, 1626, 1590, 1519, 1484, 1445, 1359, 1275, 1017, 816, 631.

35 - HRMS (ESI): calcd. for C16H5 Cl4F2N2O2 (M-H) 434.90787; found 434.90791.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

130 EXPERIMENTAL PART

N-(4-Bromophenyl)-2-cyano-3-(3,5-dibromophenyl)-3-hydroxyacrylamide – SGA-75 (68)

According to general procedure B, amide 25 (239 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dibromobenzoic acid (308 mg, 1.10 mmol, 1.1 eq.; converted into the corresponding aryl chloride) were used to give SGA-75 (68) as colorless crystals (222 mg, 0.443 mmol, 44%).

Rf = 0.20 (3:2 hexanes/acetone). m.p.: 233 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.05 (s, 1H, NH), 7.86 (t, J = 1.8 Hz, 1H, 4’-H), 7.73 (d, J = 1.8 Hz, 2H, 2’’-H, 6’’-H), 7.57 – 7.50 (m, 2H, 2’-H, 6’-H), 7.46 – 7.37 (m, 2H, 3’-H, 5’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 182.2 (C-3), 166.1 (C-1), 145.5 (C-1’’), 139.4 (C-1’), 133.7 (C-4’’), 131.5 (C-3’, C-5’), 129.2 (C-2’’, C-6’’), 122.7 (C-2), 121.8 (C-3’’, C-5’’), 120.8 (C- 2’, C-6’), 113.0 (C-4’), 77.4 (CN).

-1 IR (ATR) ṽmax/cm = 3314, 2214, 1596, 1543, 865, 817, 748, 658.

79 - HRMS (ESI): calcd. for C16H8 Br3N2O2 (M-H) 496.81414; found 496.81449.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

N-(4-Acetylphenyl)-2-cyano-3-(3,5-dichlorophenyl)-3-hydroxyacrylamide – SGA-76 (69)

According to general procedure B, amide 30 (202 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-76 (69) as off white solid (296 mg, 0.789 mmol, 79%).

Rf = 0.08 (3:2 hexanes/acetone). m.p.: 209 °C.

131 EXPERIMENTAL PART

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 12.32 (s, 1H, NH), 7.91 – 7.86 (m, 2H, 3’-H, 5’-H), 7.71 – 7.66 (m, 2H, 2’-H, 6’-H), 7.64 (t, J = 1.9 Hz, 1H, 4’’-H), 7.58 (d, J = 1.9 Hz, 2H, 2’’-H, 6’’-H),

2.51 (s, 3H, CH3, collapses with DMSO).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 196.3 (CO), 182.8 (C-3), 166.3 (C-1), 145.1 (C-1’’), 144.5 (C-1’), 133.5 (C-3’’, C-5’’), 130.4 (C-4’), 129.7 (C-3’, C-5’), 128.5 (C-4’’), 126.1 (C-2’’, C-

6’’), 123.3 (C-2), 117.9 (C-2’, C-6’), 77.6 (CN), 26.3 (CH3).

-1 IR (ATR) ṽmax/cm = 3304, 2207, 1682, 1596, 1544, 1355, 1272, 809.

35 - HRMS (ESI): calcd. for C18H11 Cl2N2O3 (M-H) 373.01522; found 373.01580.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-N-(2,6-dibromophenyl)-3-(3,5-dichlorophenyl)-3-hydroxyacrylamide – SGA-77 (70)

According to general procedure B, amide 39 (318 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-77 (70) as colorless crystals (66.0 mg, 0.134 mmol, 13%).

Rf = 0.78 (3:2 hexanes/acetone). m.p.: 226 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 10.64 (s, 1H, NH), 8.09 – 7.90 (m, 3H, 2’’-H, 6’’-H, 4’’- H), 7.79 (d, J = 8.0 Hz, 2H, 3’-H, 5’-H), 7.26 (t, J = 8.0 Hz, 1H, 4’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 162.4 (C-1, C-3), 136.6 (C-1’’), 135.2 (C-1’), 134.6 (C- 3’’, C-5’’), 132.3 (C-3’, C-5’), 131.5 (C-4’’), 130.7 (C-4’), 126.4 (C-2’’, C-6’’), 124.4 (C-2’, C-6’), 76.4 (CN), C-2 is missing.

-1 IR (ATR) ṽmax/cm = 3206, 2215, 1650.1567, 1516, 1283, 779, 750, 723.

79 35 - HRMS (ESI): calcd. for C16H7 Br2 Cl2N2O2 (M-H) 486.82568; found 486.82870.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

132 EXPERIMENTAL PART

2-Cyano-N-(2,3-dichlorophenyl)-3-(3,5-dichlorophenyl)-3-hydroxyacrylamide – SGA-78 (71)

According to general procedure B, amide 38 (229 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-78 (71) as colorless crystals (344 mg, 0.856 mmol, 86%).

Rf = 0.17 (3:2 hexanes/acetone). m.p.: 211 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.49 (s, 1H, NH), 8.58 (dd, J = 8.3, 1.5 Hz, 1H, 6’-H), 7.63 (t, J = 1.9 Hz, 1H, 4’’-H), 7.59 (d, J = 1.9 Hz, 2H, 2’’-H, 6’’-H), 7.27 (t, J = 8.2 Hz, 1H, 5’- H), 7.20 (dd, J = 8.0, 1.5 Hz, 1H, 4’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 182.8 (C-3), 166.4 (C-1), 145.1 (C-1’’), 139.3 (C-1’), 133.4 (C-3’’, C-5’’), 131.4 (C-3’), 128.5 (C-4’’), 128.0 (C-5’), 126.1 (C-2’’, C-6’’), 123.4 (C-2), 122.6 (C-4’), 119.3 (C-2’), 119.1 (C-6’), 77.5 (CN).

-1 IR (ATR) ṽmax/cm = 3355, 2203, 1649, 1584, 1539, 1453, 1415, 872, 812, 777.

35 - HRMS (ESI): calcd. for C16H7 Cl4N2O2 (M-H) 398.92671; found 398.92789.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dichlorophenyl)-3-hydroxy-N-(4-propoxyphenyl)acrylamide – SGA-84 (72)

According to general procedure B, amide 31 (218 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-84 (72) as yellow solid (245 mg, 0.626 mmol, 63%).

Rf = 0.15 (3:2 hexanes/acetone + 2% TEA).

133 EXPERIMENTAL PART m.p.: 184 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 11.36 (s, 1H, NH), 9.30 (s, 1H, OH), 7.71 (t, J = 1.7 Hz, 1H, 4’’-H), 7.67 – 7.62 (m, 2H, 2’’-H, 6’’-H), 7.49 – 7.40 (m, 2H, 3’-H, 5’-H), 6.90 – 6.82 (m,

2H, 2’-H, 6’-H), 3.88 (t, J = 6.5 Hz, 2H, CH2CH2CH3), 1.71 (sext, J = 7.2 Hz, 2H, CH2CH2CH3),

0.97 (t, J = 7.4 Hz, 3H, CH2CH2CH3).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 181.6 (C-3), 166.4 (C-1), 154.6 (C-4’), 142.7 (C-1’’), 133.7 (C-3’’, C-5’’), 131.9 (C-1’), 129.3 (C-4’’), 126.3 (C-2’’, C-6’’), 121.6 (C-3’, C-5’), 121.5 (C-

2), 114.5 (C-2’, C-6’), 78.0 (CN), 69.1 (CH2CH2CH3), 22.1 (CH2CH2CH3), 10.4 (CH2CH2CH3).

-1 IR (ATR) ṽmax/cm = 3304, 2208, 1601, 1550, 1511, 1249, 1235, 822, 811.

35 - HRMS (ESI): calcd. for C19H15 Cl2N2O3 (M-H) 389.04652; found 389.04631.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

N-(3,5-Bis(trifluoromethyl)phenyl)-2-cyano-3-(3,5-dichlorophenyl)-3-hydroxyacrylamide – SGA-85 (73)

According to general procedure B, amide 40 (296 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-85 (73) as colorless solid (320 mg, 0.682 mmol, 68%).

Rf = 0.15 (3:2 hexanes/acetone). m.p.: 210 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.58 (s, 1H, NH), 8.93 (s, 1H, OH), 8.25 (s, 2H, 2’-H, 6’-H), 7.65 (t, J = 1.9 Hz, 1H, 4’’-H), 7.62 – 7.56 (m, 3H, 4’-H, 2’’-H, 6’’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 183.5 (C-3), 167.3 (C-1), 145.4 (C-1’’), 142.3 (C-1’),

134.0 (C-3’’, C-5’’), 131.2 (q, JCF = 32.6 Hz, C-3’, C-5’), 129.1 (C-4’’), 126.5 (C-2’’, C-6’’), 123.7

(q, JCF = 270.8 Hz, CF3), 123.4 (C-2), 119.1 – 118.8 (m, C-2’, C-6’), 114.8 – 114.5 (m, C-4’), 77.6 (CN).

-1 IR (ATR) ṽmax/cm = 2230, 1637, 1571, 1547, 1375, 1275, 1175, 1128, 810.

134 EXPERIMENTAL PART

35 - HRMS (ESI): calcd. for C18H7 Cl2F6N2O2 (M-H) 466.97943; found 466.97933.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dichlorophenyl)-3-hydroxy-N-(4-(trifluoromethoxy)phenyl)acrylamide – SGA-86 (74)

According to general procedure B, amide 32 (244 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-86 (74) as colorless solid (344 mg, 0.825 mmol, 83%).

Rf = 0.53 (3:2 hexanes/acetone). m.p.: 195 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 12.02 (s, 1H, NH), 9.41 (s, 1H, OH), 7.69 – 7.64 (m, 3H, 2’-H, 6’-H, 4’’-H), 7.59 (d, J = 1.9 Hz, 2H, 2’’-H, 6’’-H), 7.28 – 7.23 (m, 2H, 3’-H, 5’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 182.3 (C-3), 166.3 (C-1), 144.6 (C-1’’), 142.7 (C-4’), 139.1 (C-1’), 133.5 (C-3’’, C-5’’), 128.6 (C-4’’), 126.1 (C-2’’, C-6’’), 123.0 (C-2), 121.6 (C-3’, C-

5’), 120.3 (q, JCF = 255.2 Hz, OCF3), 120.2 (C-2’, C-6’), 77.6 (CN).

-1 IR (ATR) ṽmax/cm = 3304, 2218, 1614, 1536, 1506, 1262, 1208, 1164, 661.

35 - HRMS (ESI): calcd. for C17H8 Cl2F3N2O3 (M-H) 414.98696; found 414.98676.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

N-(3-Chloro-2,4-difluorophenyl)-2-cyano-3-(3,5-dibromophenyl)-3-hydroxyacrylamide – SGA-90 (75)

135 EXPERIMENTAL PART

According to general procedure B, amide 36 (231 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dibromobenzoic acid (308 mg, 1.10 mmol, 1.1 eq.; converted into the corresponding aryl chloride) were used to give SGA-90 (75) as light yellow solid (374 mg, 0.759 mmol, 76%).

Rf = 0.16 (1:1 hexanes/acetone). m.p.: 192 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.33 (s, 1H, NH), 8.43 (td, J = 8.9, 6.0 Hz, 1H, 6’-H), 7.86 (t, J = 1.7 Hz, 1H, 4’’-H), 7.74 (d, J = 1.7 Hz, 2H, 2’’-H, 6’’-H), 7.23 (td, J = 9.3, 2.0 Hz, 1H, 5’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 182.7 (C-3), 166.3 (C-1), 152.1 (d, JCF = 242.4 Hz, C-

2’), 147.7 (d, JCF = 242.4 Hz, C-4’), 145.4 (C-1’’), 133.7 (C-4’’), 129.2 (C-2’’, C-6’’), 126.3 (dd,

JCF = 10.3, 3.2 Hz, C-1’), 123.2 (C-2), 121.8 (C-3’’, C-5’’), 119.1 (dd, JCF = 7.5, 2.9 Hz, C-6’),

111.5 (dd, JCF = 20.7, 3.6 Hz, C-5’), 108.3 – 107.7 (m, C-3’), 77.3 (CN).

-1 IR (ATR) ṽmax/cm = 3297, 2205, 1586, 1531, 1496, 1022, 803, 750.

79 - HRMS (ESI): calcd. for C16H6 Br2ClF2N2O2 (M-H) 488.84581; found 488.84607.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-hydroxy-N-(4-(trifluoromethyl)phenyl)but-2-enamide (teriflunomide) – SGA-94 (76)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and acetyl chloride (78.5 µL, 1.10 mmol, 1.1 eq.) were used to give SGA-94 (76) as colorless crystals (206 mg, 0.761 mmol, 76%). Analytical data are in accordance with literature.[189]

Rf = 0.65 (3:2 hexanes/acetone). m.p.: 224 °C. [lit.[189]: 230 – 232 °C]

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.28 (s, 1H, OH), 10.91 (s, 1H, NH), 7.81 – 7.72 (m, 2H, 2’-H, 6’-H), 7.70 – 7.61 (m, 2H, 3’-H, 5’-H), 2.25 (s, 3H, 4-H).

136 EXPERIMENTAL PART

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 187.1 (C-3), 166.4 (C-1), 141.9 (C-1’), 125.9 (q, JCF =

3.7 Hz, C-3’, C-5’), 124.4 (q, JCF = 271.3 Hz, CF3), 123.5 (q, JCF = 32.1 Hz, C-4’), 120.7 (C-2’, C-6’), 118.9 (C-2), 80.5 (CN), 23.5 (C-4).

-1 IR (ATR) ṽmax/cm = 2335, 2214, 1551, 1319, 1154, 1113, 840, 679.

- HRMS (ESI): calcd. for C12H8F3N2O2 (M-H) 269.05434; found 269.05423.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

3-(3,5-Bis(trifluoromethyl)phenyl)-2-cyano-3-hydroxy-N-(p-tolyl)acrylamide – SGA-108 (77)

According to general procedure B, amide 21 (174 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-bis(trifluoromethyl)benzoyl chloride (199 µL, 1.10 mmol, 1.1 eq.) were used to give SGA-108 (77) as light yellow crystals (208 mg, 0.502 mmol, 50%).

Rf = 0.28 (3:2 hexanes/acetone). m.p.: 183 °C.

1 H NMR (500 MHz, CDCl3) δ/ppm = 8.45 (s, 2H, 2’’-H, 6’’-H), 8.10 (s, 1H, 4’’-H), 7.86 (s, 1H,

NH), 7.43 – 7.37 (m, 2H, 2’-H, 6’-H), 7.25 – 7.20 (m, 2H, 3’-H, 5’-H), 2.37 (s, 3H, CH3).

13 C NMR (126 MHz, CDCl3) δ/ppm = 180.4 (C-3), 167.9 (C-1), 136.7 (C-4’), 134.7 (C-1’’), 132.7

(C-1’), 132.6 (q, JCF = 34.2 Hz, C-3’’, C-5’’), 130.1 (C-3’, C-5’), 128.6 (q, JCF = 3.1 Hz, C-2’’, C-

6’’), 126.3 (q, JCF = 3.7 Hz, C-4’’), 122.8 (q, JCF = 272.9 Hz, CF3), 121.8 (C-2’, C-6’), 116.5 (C-

2), 79.7 (CN), 21.2 (CH3).

-1 IR (ATR) ṽmax/cm = 3276, 2213, 1538, 1278, 1129, 811, 681.

- HRMS (ESI): calcd. for C19H11F6N2O2 (M-H) 413.07302; found 413.07301.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

137 EXPERIMENTAL PART

3-(3,5-Bis(trifluoromethyl)phenyl)-2-cyano-3-hydroxy-N-(4-(trifluoromethyl)phenyl)- acrylamide – SGA-111 (78)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-bis(trifluoromethyl)benzoyl chloride (199 µL, 1.10 mmol, 1.1 eq.) were used to give SGA-111 (78) as colorless crystals (357 mg, 0.762 mmol, 76%).

Rf = 0.43 (3:2 hexanes/acetone). m.p.: 230 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.36 (s, 1H, NH), (m, 3H, 2’’-H, 6’’-H, 4’’-H), 7.87 – 7.71 (m, 2H, 2’-H, 6’-H), 7.69 – 7.51 (m, 2H, 3’-H, 5’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 182.4 (C-3), 166.3 (C-1), 144.0 (C-1’’), 143.6 (C-1’),

129.8 (q, JCF = 32.8 Hz, C-3’’, C-5’’), 128.1 (q, JCF = 4.7 Hz, C-2’’, C-6’’), 126.1 (q, JCF = 3.6

Hz, C-3’, C-5’), 126.0 (q, JCF = 257.2 Hz, m-CF3), 123.4 (C-2), 123.3 (q, JCF = 271.6 Hz, p-CF3),

122.7 (q, JCF = 4.0 Hz, C-4’’), 121.7 (q, JCF = 31.9 Hz, C-4’), 118.5 (C-2’, C-6’), 77.7 (CN).

-1 IR (ATR) ṽmax/cm = 3289, 2218, 1349, 1323, 1284, 1186, 1139, 1114, 836.

- HRMS (ESI): calcd. for C19H8F9N2O2 (M-H) 467.04475; found 467.04496.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dimethylphenyl)-3-hydroxy-N-(4-(trifluoromethyl)phenyl)acrylamide – SGA-112 (79)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dimethylbenzoyl chloride (163 µL,

138 EXPERIMENTAL PART

1.10 mmol, 1.1 eq.) were used to give SGA-112 (79) as colorless crystals (175 mg, 0.486 mmol, 49%).

Rf = 0.48 (3:2 hexanes/acetone). m.p.: 187 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 12.08 (s, 1H, NH), 7.85 – 7.74 (m, 2H, 2’-H, 6’-H), 7.66

– 7.58 (m, 2H, 3’-H, 5’-H), 7.25 (s, 2H, 2’’-H, 6’’-H), 7.09 (s, 1H,4’’-H), 2.30 (s, 6H, CH3).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 186.5 (C-3), 167.8 (C-1), 143.6 (C-1’), 140.1 (C-1’’),

137.2 (C-3’’, C-5’’), 131.8 (C-4’’), 126.4 (q, JCF = 3.8 Hz, C-3’, C-5’), 125.8 (C-2’’, C-6’’), 125.3

(q, JCF = 270.8 Hz, CF3), 122.6 (q, JCF = 31.9 Hz, C-4’), 121.8 (C-2), 119.9 (C-2’, C-6’), 78.2

(CN), 21.4 (CH3).

-1 IR (ATR) ṽmax/cm = 3270, 2218, 1526.1319, 1247, 1157, 1110, 1066, 839.

- HRMS (ESI): calcd. for C19H14F3N2O2 (M-H) 359.10129; found 359.10142.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dimethoxyphenyl)-3-hydroxy-N-(4-(trifluoromethyl)phenyl)acrylamide – SGA-113 (80)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dimethoxybenzoyl chloride (221 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-113 (80) as colorless crystals (309 mg, 0.789 mmol, 79%).

Rf = 0.54 (3:2 hexanes/acetone). m.p.: 203 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.99 (s, 1H, NH), 7.73 – 7.63 (m, 4H, 2’-H, 6’-H, 3’-H, 5’- 1 H), 7.12 (d, J = 2.3 Hz, 2H, 2’’-H, 6’’-H), 6.69 (t, J = 2.3 Hz, 1H, 4’’-H), 3.85 (s, 6H, OCH3).

3 C NMR (101 MHz, CDCl3) δ/ppm = 184.1 (C-3), 168.7 (C-1), 161.0 (C-3’’, C-5’’), 139.1 (C-1’),

133.9 (C-1’’), 127.8 (q, JCF = 33.2 Hz, C-4’), 126.7 (q, JCF = 3.8 Hz, C-3’, C-5’), 123.9 (q, JCF =

139 EXPERIMENTAL PART

270.1 Hz, CF3), 121.0 (C-2’, C-6’), 117.5 (C-2), 106.4 (C-2’’, C-6’’), 106.0 (C-4’’), 78.5 (CN),

55.8 (OCH3).

-1 IR (ATR) ṽmax/cm = 3297, 2215, 1550, 1324, 1208, 1156, 1095, 1067, 834.

- HRMS (ESI): calcd. for C19H14F3N2O4 (M-H) 391.09112; found 391.09140.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-hydroxy-3-(4-(trifluoromethoxy)phenyl)-N-(4-(trifluoromethyl)phenyl)- acrylamide – SGA-114 (81)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 4-(trifluoromethoxy)benzoyl chloride (173 µL, 1.10 mmol, 1.1 eq.) were used to give SGA-114 (81) as colorless crystals (200 mg, 0.481 mmol, 48%).

Rf = 0.58 (3:2 hexanes/acetone). m.p.: 198 °C. [lit.[91]: 188 – 190 °C]

1 H NMR (500 MHz, CDCl3) δ/ppm = 8.12 – 8.05 (m, 2H, 2’’-H, 6’’-H), 7.96 (s, 1H, NH), 7.78 – 7.62 (m, 4H, 2’-H, 6’-H, 3’-H, 5’-H), 7.42 – 7.32 (m, 2H, 3’’-H, 5’’-H).

13 C NMR (126 MHz, CDCl3) δ/ppm = 182.6 (C-3), 168.5 (C-1), 152.8 (C-4’’), 139.0 (C-1’), 130.7

(C-2’’, C-6’’), 130.5 (C-1’’), 128.0 (q, JCF = 33.0 Hz, C-4’), 126.7 (q, JCF = 3.7 Hz, C-3’, C-5’),

123.9 (q, JCF = 271.7 Hz, CF3), 121.0 (C-2’, C-6’), 120.8 (C-3’’, C-5’’), 120.6 (q, JCF = 259.4 Hz,

OCF3), 117.3 (C-2), 78.6 (CN).

-1 IR (ATR) ṽmax/cm = 3285, 2215, 1597, 1551, 1505, 1268, 1168, 1128, 839.

- HRMS (ESI): calcd. for C18H9F6N2O3 (M-H) 415.05228; found 415.05225.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

140 EXPERIMENTAL PART

2-Cyano-3-(3,5-dichlorophenyl)-3-hydroxy-N-methyl-N-(4-(trifluoromethyl)phenyl)- acrylamide – SGA-115 (82)

According to general procedure B, amide 41 (242 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-115 (82) as colorless solid (198 mg, 0.476 mmol, 48%).

Rf = 0.67 (3:2 hexanes/acetone). m.p.: 147 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.80 – 7.73 (m, 2H, 3’-H, 5’-H), 7.64 (d, J = 1.9 Hz, 2H,

2’’-H, 6’’-H), 7.49 (t, J = 1.9 Hz, 1H, 4’’-H), 7.47 – 7.41 (m, 2H, 2’-H, 6’-H), 3.48 (s, 3H, CH3).

13 C NMR (101 MHz, CDCl3) δ/ppm = 184.2 (C-3), 170.4 (C-1), 145.2 (C-1’), 135.8 (C-1’’), 135.5

(C-3’’, C-5’’), 132.5 (C-4’’), 131.4 (q, JCF = 32.9 Hz, C-4’), 127.8 (C-2’, C-6’), 127.5 (q, J = 3.6

Hz, C-3’, C-5’), 127.2 (C-2’’, C-6’’), 123.7 (q, JCF = 272.6 Hz, CF3), 115.3 (C-2), 78.6 (CN), 39.9

(CH3).

-1 IR (ATR) ṽmax/cm = 3074, 2214, 1578, 1540, 1396, 1331, 1165, 1118, 807.

35 - HRMS (ESI): calcd. for C18H10 Cl2F3N2O2 (M-H) 413.00769; found 413.00806.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-hydroxy-3-(pyridin-3-yl)-N-(4-(trifluoromethyl)phenyl)acrylamide – SGA-127 (83)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and nicotinoyl chloride hydrochloride (196 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-127 (83) as orange solid (169 mg, 0.506 mmol, 51%).

141 EXPERIMENTAL PART

Rf = 0.00 (3:2 hexanes/EtOAc). m.p.: 235 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.22 (s, 1H, NH), 9.12 (s, 1H, 2’’-H), 8.88 (d, J = 5.6 Hz, 1H, 6’’-H), 8.70 (d, J = 8.0 Hz, 1H, 4’’-H), 8.04 (dd, J = 8.0, 5.6 Hz, 1H, 5’’-H), 7.83 – 7.72 (m, 2H, 2’-H, 6’-H), 7.67 – 7.56 (m, 2H, 3’-H, 5’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 180.0 (C-3), 165.9 (C-1), 143.5 (C-1’), 143.3 (C-6’’),

143.0 (C-4’’), 141.9 (C-2’’), 139.9 (C-3’’), 126.2 (C-5’’), 126.1 (q, JCF = 3.6 Hz, C-3’, C-5’), 124.2

(q, JCF = 270.7 Hz, CF3), 123.3 (C-2), 121.8 (q, JCF = 32.0 Hz, C-4’), 118.5 (C-2’, C-6’), 78.5 (CN).

-1 IR (ATR) ṽmax/cm = 2356, 2191, 1533, 1317, 1105, 1060, 849, 698.

- HRMS (ESI): calcd. for C16H9F3N3O2 (M-H) 332.06523; found 332.06517.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

3-(3-Bromo-5-iodophenyl)-2-cyano-3-hydroxy-N-(4-(trifluoromethyl)phenyl)acrylamide – SGA-132 (84)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3-bromo-5-iodobenzoic acid (360 mg, 1.10 mmol, 1.1 eq.; converted into the corresponding aryl chloride) were used to give SGA- 132 (84) as colorless crystals (332 mg, 0.618 mmol, 62%).

Rf = 0.38 (3:2 hexanes/EtOAc). m.p.: 207 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.31 (s, 1H, NH), 7.97 (s, 1H, 4’’-H), 7.89 (s, 1H, 2’’- H or 6’’-H), 7.78 – 7.72 (m, 3H, 2’-H, 6’-H and 2’’-H or 6’’-H), 7.63 – 7.55 (m, 2H, 3’-H, 5’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 182.6 (C-3), 166.4 (C-1), 145.4 (C-1’’), 143.6 (C-1’),

139.1 (C-4’’), 135.0 (C-2’’ or C-6’’), 129.5 (C-2’’ or C-6’’), 126.0 (q, JCF = 3.8 Hz, C-3’, C-5’),

123.9 (q, JCF = 263.8 Hz, CF3), 123.3 (C-2), 121.7 (q, JCF = 31.6 Hz, C-4’), 121.6 (C-3’’ or C- 5’’), 118.6 (C-2’, C-6’), 95.0 (C-3’’ or C-5’’), 77.4 (CN).

142 EXPERIMENTAL PART

-1 IR (ATR) ṽmax/cm = 3276, 2213, 1532, 1318, 1163, 1112, 731.

79 - HRMS (ESI): calcd. for C17H8 BrIF3N2O2 (M-H) 534.87714; found 534.87813.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

Methyl 4-(2-cyano-3-(3,5-dichlorophenyl)-3-hydroxyacrylamido)benzoate – SGA-133 (85)

According to general procedure B, amide 33 (218 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used. The resulting solid was washed with hexanes, EtOH and water to give SGA-133 (85) as colorless solid (331 mg, 0.846 mmol, 85%).

Rf = 0.15 (3:2 hexanes/acetone). m.p.: 234 °C.

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 12.33 (s, 1H, NH), 11.41 (s, 1H, OH), 7.90 – 7.83 (m, 2H, 2-H, 6-H), 7.71 – 7.66 (m, 2H, 3-H, 5-H), 7.64 (t, J = 1.9 Hz, 1H, 4’’-H), 7.57 (d, J = 1.9 Hz,

2H, 2’’-H, 6’’-H), 3.81 (s, 3H, CH3).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 182.8 (C-3’), 166.3 (C-1’), 166.0 (CO), 145.1 (C-1’’), 144.6 (C-4), 133.4 (C-3’’, C-5’’), 130.4 (C-2, C-6), 128.5 (C-4’’), 126.1 (C-2’’, C-6’’), 123.3 (C-

2’), 122.3 (C-1), 118.1 (C-3, C-5), 77.6 (CN), 51.7 (CH3).

-1 IR (ATR) ṽmax/cm = 3304, 3093, 2215, 1727, 1591, 1534, 1415, 1283, 1262, 1112, 810, 766.

35 - HRMS (ESI): calcd. for C18H11 Cl2N2O4 (M-H) 389.01014; found 389.01077.

Purity (NMR): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

143 EXPERIMENTAL PART

3-(5-Chloropyridin-3-yl)-2-cyano-3-hydroxy-N-(4-(trifluoromethyl)phenyl)acrylamide – SGA-136 (86)

According to general procedure B, SGA-34 (19, 228 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 5-chloronicotinic acid (173 mg, 1.10 mmol, 1.1 eq.; converted into the corresponding aryl chloride) were used to give SGA-136 (86) as light pink crystals (225 mg, 0.611 mmol, 61%).

Rf = 0.15 (3:2 hexanes/acetone). m.p.: 221 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 12.33 (s, 1H, NH), 8.74 (s, 1H, 2’’-H or 4’’-H), 8.69 – 8.64 (m, 1H, 6’’-H), 8.11 – 8.05 (m, 1H, 2’’-H or 4’’-H), 7.81 – 7.73 (m, 2H, 2’-H, 6’-H), 7.64 – 7.55 (m, 2H, 3’-H, 5’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 181.7 (C-3), 166.2 (C-1), 147.9 (C-6’’), 146.1 (C-2’’ or C-4’’), 143.6 (C-1’), 138.8 (C-3’’ or C-5’’), 134.9 (C-2’’ or C-4’’), 130.5 (C-3’’ or C-5’’), 126.1 (q,

JCF = 3.7 Hz, C-3’, C-5’), 124.6 (q, JCF = 271.0 Hz, CF3), 123.5 (C-2), 121.6 (q, JCF = 31.5 Hz, C-4’), 118.5 (C-2’, C-6’), 78.2 (CN).

-1 IR (ATR) ṽmax/cm = 3285, 2225, 1539, 1308, 1113, 951, 838.

35 - HRMS (ESI): calcd. for C16H8 ClF3N3O2 (M-H) 366.02626; found 366.02652.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

4-(2-Cyano-3-(3,5-dichlorophenyl)-3-hydroxyacrylamido)benzoic acid – SGA-137 (88)

Ester 85 (95.0 mg, 0.243 mmol, 1.0 eq.) was dissolved in dioxane/H2O (3:1; 4.0 mL) and LiOH (61.2 mg, 2.43 mmol, 10 eq.) was added. The mixture was stirred at rt for 3 h, before 1 M aq.

144 EXPERIMENTAL PART

HCl (5.0 mL) was added. The precipitate was filtered off, washed with cold water and dried to give SGA-137 (88) as colorless solid (59.6 mg, 0.158 mmol, 65%).

Rf = 0.00 (3:2 hexanes/acetone). m.p.: 244 °C.

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 12.30 (s, 1H, NH), 7.87 – 7.81 (m, 2H, 2-H, 6-H), 7.68 – 7.64 (m, 2H, 3-H, 5-H), 7.63 (t, J = 1.9 Hz, 1H, 4’’-H), 7.57 (d, J = 1.9 Hz, 2H, 2’’-H, 6’’-H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 182.7 (C-3’), 167.1 (CO), 166.3 (C-1’), 145.2 (C-1’’), 144.2 (C-4), 133.4 (C-3’’, C-5’’), 130.5 (C-2, C-6), 128.4 (C-4’’), 126.1 (C-2’’, C-6’’), 123.5 (C- 1), 123.4 (C-2’), 118.0 (C-3, C-5), 77.5 (CN).

-1 IR (ATR) ṽmax/cm = 3311, 2215, 1696, 1595, 1550, 1415, 1294, 855, 770.

35 - HRMS (ESI): calcd. for C17H9 Cl2N2O4 (M-H) 374.99449; found 374.99480.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dichlorophenyl)-3-hydroxy-N-(4-(trifluoromethyl)benzyl)acrylamide – SGA-138 (87)

According to general procedure B, amide 42 (242 mg, 1.00 mmol, 1.0 eq.) in dry THF (10 mL), NaH (92.0 mg, 2.30 mmol, 2.3 eq.) and 3,5-dichlorobenzoyl chloride (43, 230 mg, 1.10 mmol, 1.1 eq.) were used to give SGA-138 (87) as colorless solid (228 mg, 0.549 mmol, 55%).

Rf = 0.70 (3:2 hexanes/acetone). m.p.: 173 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.80 – 7.73 (m, 2H, 3’-H, 5’-H), 7.64 (d, J = 1.9 Hz, 2H,

2’’-H, 6’’-H), 7.49 (t, J = 1.9 Hz, 1H, 4’’-H), 7.47 – 7.41 (m, 2H, 2’-H, 6’-H), 3.48 (s, 3H, CH3).

13 C NMR (101 MHz, CDCl3) δ/ppm = 184.2 (C-3), 170.4 (C-1), 145.2 (C-1’), 135.8 (C-1’’), 135.5

(C-3’’, C-5’’), 132.5 (C-4’’), 131.4 (q, JCF = 32.9 Hz, C-4’), 127.8 (C-2’, C-6’), 127.5 (q, J = 3.6

Hz, C-3’, C-5’), 127.2 (C-2’’, C-6’’), 123.7 (q, JCF = 272.6 Hz, CF3), 115.3 (C-2), 78.6 (CN), 39.9

(CH3).

-1 IR (ATR) ṽmax/cm = 3074, 2214, 1578, 1540, 1396, 1331, 1165, 1118, 807.

145 EXPERIMENTAL PART

35 - HRMS (ESI): calcd. for C18H10 Cl2F3N2O2 (M-H) 413.00769; found 413.00806.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Cyano-3-(3,5-dichlorophenyl)-3-hydroxy-N-(4-(trifluoromethyl)phenyl)prop-2- enethioamide – SGA-167 (91)

According to a procedure published by Sjogren, et al. [91], a stirred solution of acetonitrile (2.01 mL, 38.2 mmol, 4.0 eq.) in THF (70 mL) was cooled to -78 °C, n-BuLi (2.5 M in hexanes, 11.5 mL, 28.6 mmol, 3.0 eq.) was added over 10 minutes and the mixture was stirred for further 15 minutes. A solution of 3,5-dichlorobenzoyl chloride (43, 2.00 g, 9.55 mmol, 1.0 eq.) in THF (25 mL) was added over 10 minutes. The mixture was allowed to warm to rt over 40 minutes, followed by addition of sat. aq. NH4Cl solution (50 mL). The reaction mixture was partitioned between 1 M HCl and hexanes/EtOAc (two times 25 mL/25 mL). The combined organic phases were dried over Na2SO4, filtered and concentrated in vacuo. The oily residue was redissolved in aq. NH3 solution (25%, 10 mL), then acidified with 1 M HCl (10 mL) and extracted thrice with EtOAc (15 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo and then recrystallized from hexanes to yield 3-(3,5- dichlorophenyl)-3-oxopropanenitrile (89) as orange solid (526 mg, 2.46 mmol, 26%). Analytical data are in accordance with literature.[190] The product was used without further purification or characterization for the next step.

Rf = 0.27 (9:1 hexanes/EtOAc).

35 + HRMS (EI): calcd. for C9H5 Cl2NO (M)˙ 212.9743; found 212.9742.

According to general procedure B, propanenitrile 89 (526 mg, 2.46 mmol, 1.0 eq, instead of an amide) in dry THF (10 mL), NaH (108 mg, 2.70 mmol, 1.1 eq.) and 4-(trifluoromethyl)phenyl isothiocyanate (90, 524 mg, 2.85 mmol, 1.1 eq., instead of the acid chloride) were used to give SGA-167 (91) as golden solid (384 mg, 0.92 mmol, 37%).

Rf = 0.33 (9:1 hexanes/EtOAc). m.p.: 188 °C.

146 EXPERIMENTAL PART

1 H NMR (400 MHz, (CD3)2SO) δ/ppm = 14.34 (s, 1H, NH), 8.12 (d, J = 8.5 Hz, 2H, 2’-H, 6’-H), 7.72 – 7.63 (m, 3H, 3’-H, 5’-H, 4’’-H), 7.57 (d, J = 1.9 Hz, 2H, 2’’-H, 6’’-H).

13 C NMR (101 MHz, (CD3)2SO) δ/ppm = 189.4 (C-1), 182.0 (C-3), 144.9 (C-2), 144.1 (C-1’),

133.4 (C-3’’, C-5’’), 128.6 (C-4’’), 126.1 (C-2’’, C-6’’), 125.5 (q, JCF = 3.7 Hz, C-3’, C-5’), 124.4

(q, JCF = 271.3 Hz, CF3), 123.8 (q, JCF = 31.9 Hz, C-4’), 123.6 (C-1’’), 122.2 (C-2’, C-6’), 90.9 (CN).

-1 IR (ATR) ṽmax/cm = 3267, 2362, 2202, 1550, 1521, 1323, 1106, 1067, 807, 671, 585.

35 - HRMS (ESI): calcd. for C17H8 Cl2F3N2OS (M-H) 414.96920; found 414.96915.

Purity (HPLC): > 96% (λ = 210 nm).

147 EXPERIMENTAL PART

5.1.2.2 Synthesis of TPC2-A1-P (17) and analogs

1-(5-Bromo-2-(trifluoromethoxy)phenyl)-2-chloroethan-1-one (102)

4-Bromo-2-iodo-1-(trifluoromethoxy)benzene (108, 610 mg, 1.66 mmol, 1.0 eq.) was dissolved in dry THF (8.0 mL) and cooled to -78 °C, then n-BuLi (0.670 mL, 1.66 mmol, 1.0 eq.) was added dropwise. The mixture was stirred for 20 min at -78 °C and a solution of 2-chloro-N- methoxy-N-methylacetamide (105, 700 mg, 4.99 mmol, 3.0 eq.) in dry THF (8.0 mL) was added slowly. The mixture was stirred for 1 h at -78 °C and then poured on sat. aq. NH4Cl solution. The mixture was extracted with pentane, the organic layer was washed with sat. aq. NaCl solution, dried using hydrophobic phase separation filter papers and filtered through a short silica column (eluent: pentane). The product was carefully concentrated under ambient pressure to yield a colorless oil (102, 221 mg, 0.696 mmol, 42%).

Rf = 0.65 (9:1 hexanes/EtOAc).

1 H NMR (500 MHz, CDCl3) δ/ppm = 7.93 (d, J = 2.5 Hz, 1H, 6’-H), 7.71 (dd, J = 8.8, 2.5 Hz, 1H, 4’-H), 7.25 – 7.22 (m, 1H, 3’-H), 4.61 (s, 2H, 2-H).

13 C NMR (126 MHz, CDCl3) δ/ppm = 190.5 (C-1), 146.2 (C-2’), 137.1 (C-4’), 134.1 (C-6’), 130.8

(C-5’), 122.2 (C-3’), 120.8 (C-1’), 120.3 (q, JCF = 261 Hz, OCF3), 49.0 (C-2).

-1 IR (ATR) ṽmax/cm = 1703, 1592, 1480, 1398, 1308, 1252, 1174, 1129, 1088, 822, 664.

79 35 + HRMS (EI): calcd. for C9H5 Br ClF3O2 (M)˙ 315.9108; found 315.9106.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

2-Bromo-1-(2-(trifluoromethoxy)phenyl)ethan-1-one (101)

2'-(Trifluoromethoxy)acetophenone (103, 779 µL, 4.90 mmol, 1.0 eq.) was dissolved in CH2Cl2 (10 mL), then p-toluenesulfonic acid (86.1 mg, 0.490 mmol, 0.10 eq.) and N-bromosuccinimide (872 mg, 4.90 mmol, 1.0 eq.) were added. The mixture was stirred for 24 h at rt, then sat. aq.

148 EXPERIMENTAL PART

NaCl solution (10 mL) was added. The aqueous phase was extracted with CH2Cl2 (3 x 10 mL), dried using a hydrophobic filter paper and concentrated in vacuo. Purification by FCC (pentane/EtOAc 9:1) yielded bromoketone 101 (672 mg, 2.87 mmol, 49%) as light brown liquid. Analytical data are in accordance with literature[106].

Rf = 0.58 (9:1 hexanes/EtOAc).

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.81 (dd, J = 7.8, 1.8 Hz, 1H, 6’-H), 7.60 (ddd, J = 8.3, 7.6, 1.8 Hz, 1H, 4’-H), 7.41 (td, J = 7.6, 1.0 Hz, 1H, 5’-H), 7.37 – 7.32 (m, 1H, 3’-H), 4.47 (s, 2H, 2-H).

13 C NMR (101 MHz, CDCl3) δ/ppm = 191.5 (C-1), 147.1 (q, J = 1.7 Hz, C-2’), 134.3 (C-4’),

131.6 (C-6’), 129.3 (C-1’), 127.3 (C-5’), 120.6 (C-3’), 120.5 (q, J = 260.1 Hz, OCF3), 35.1 (C- 2).

-1 IR (ATR) ṽmax/cm = 1698, 1603, 1450, 1295, 1248, 1200, 1160.

79 + HRMS (EI): calcd. for C9H6 BrF3O2 (M)˙ 281.9498; found 281.9494.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

Ethyl 5-(5-bromo-2-(trifluoromethoxy)phenyl)-1-(cyclohexylmethyl)-2-methyl-1H- pyrrole-3-carboxylate – SGA-140 (109)

Following general procedure C, ethyl acetoacetate (94, 88.0 µL, 0.693 mmol, 1.1 eq.) in dry THF (3.0 mL), NaH (37.8 mg, 0.945 mmol, 1.5 eq.) and a solution of ketone 102 (200 mg, 0.630 mmol, 1.0 eq.) and KI (209 mg, 1.26 mmol, 2.0 eq.) in dry THF (3.0 mL) was used. Then, the residue was dissolved in acetic acid (6.0 mL) and cyclohexanemethanamine (95, 160 µL, 1.26 mmol, 2.0 eq.) was added. FCC (hexanes/EtOAc 99:1) yielded SGA-140 (109) as colorless oil (140 mg, 0.287 mmol, 46%).

Rf = 0.30 (9:1 hexanes/EtOAc).

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.54 – 7.50 (m, 2H, 4’-H, 6’-H), 7.20 (ddt, J = 7.6, 3.0, 1.5

Hz, 1H, 3’-H), 6.55 (s, 1H, 4-H), 4.27 (q, J = 7.1 Hz, 2H, CH2CH3), 3.59 (d, J = 7.1 Hz, 2H, CH2-

149 EXPERIMENTAL PART

cy), 2.59 (s, 3H, CH3), 1.60 – 1.54 (m, 3H, cy), 1.38 – 1.32 (m, 6H, CH2CH3, cy), 1.07 – 0.99 (m, 3H, cy), 0.68 – 0.59 (m, 2H, cy).

13 C NMR (121 MHz, CDCl3) δ/ppm = 165.6 (COOEt), 146.3 (C-2’), 137.6 (C-3), 135.7 (C-6’),

132.4 (C-4’), 129.0 (C-5’), 126.5 (C-5), 122.1 (C-3’), 120.3 (q, JCF = 260.2 Hz, OCF3), 119.9

(C-1’), 112.6 (C-2), 112.2 (C-4), 59.6 (CH2CH3), 50.8 (CH2-cy), 39.0 (cy), 30.6 (cy), 26.2 (cy),

25.7 (cy), 14.7 (CH2CH3), 12.1 (CH3).

-1 IR (ATR) ṽmax/cm = 2976, 2925, 2854, 1699, 1254, 1240, 1206, 1190, 1169, 1080, 1064, 774.

79 + HRMS (ESI): calcd. for C22H26 BrF3NO3 (M+H) 488.10427; found 488.10459.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

5-(5-Bromo-2-(trifluoromethoxy)phenyl)-1-(cyclohexylmethyl)-2-methyl-1H-pyrrole-3- carboxylic acid – TPC2-A1-P (17)

According to general procedure D, LiOH (51.6 mg, 2.05 mmol, 10 eq.) and a solution of SGA-

140 (140, 100 mg, 0.205 mmol, 1.0 eq.) in dioxane/H2O (3.0 mL) were used. After 2 h the reaction was completed and recrystallization from EtOH gave TPC2-A1-P (17) as colorless solid (51.2 mg, 0.111 mmol, 54%).

Rf = 0.14 (9:1 hexanes/EtOAc). m.p.: 202 °C.

1 H NMR (500 MHz, CDCl3) δ/ppm = 11.34 (s, 1H, COOH), 7.56 – 7.51 (m, 2H, 3’-H, 4’-H), 7.23

– 7.19 (m, 1H, 6’-H), 6.61 (s, 1H, 4-H), 3.60 (d, J = 7.1 Hz, 2H, CH2-cy), 2.60 (s, 3H, CH3), 1.62 – 1.56 (m, 3H, cy), 1.40 – 1.33 (m, 3H, cy), 1.09 – 1.01 (m, 3H, cy), 0.68 – 0.61 (m, 2H, cy).

13 C NMR (126 MHz, CDCl3) δ/ppm = 170.0 (COOH), 146.4 (C-2’), 138.8 (C-2), 135.7 (C-6’),

132.6 (C-4’), 128.8 (C-5’), 126.8 (C-5), 122.2 (C-3’), 120.3 (q, JCF = 259.3 Hz, OCF3), 119.9

(C-1’), 112.9 (C-4), 111.6 (C-3), 50.9 (CH2-cy), 39.0 (cy), 30.6 (cy), 26.2 (cy), 25.7 (cy), 12.3

(CH3).

150 EXPERIMENTAL PART

-1 IR (ATR) ṽmax/cm = 2961, 2924, 2875, 2853, 2359, 2342, 1667, 1266, 1243, 1212, 1198, 1171, 925, 779, 658.

79 - HRMS (ESI): calcd. for C20H20 BrF3NO3 (M-H) 458.05841; found 458.05889.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

Ethyl 1-(cyclohexylmethyl)-2-methyl-5-phenyl-1H-pyrrole-3-carboxylate – SGA-43 (96)

Following general procedure C, ethyl acetoacetate (94, 42.0 µL, 3.30 mmol, 1.1 eq.) in dry THF (12 mL), NaH (180 mg, 4.50 mmol, 1.5 eq.) and a solution of 2-bromo-1-phenylethan-1- one (92, 0.405 mL, 3.00 mmol, 1.0 eq.) and KI (996 mg, 6.00 mmol, 2.0 eq.) in dry THF (10 mL) were used. Then, the residue was dissolved in acetic acid (10 mL) and cyclohexanemethanamine (95, 781 µL, 6.00 mmol, 2.0 eq.) was added. FCC (hexanes/EtOAc 9:1) yielded SGA-43 (96) as colorless solid (516 mg, 1.58 mmol, 53%). The compound is literature known, but no analytical data are available[103].

Rf = 0.49 (9:1 hexanes/EtOAc). m.p.: 91 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.42 – 7.36 (m, 2H, Ph), 7.35 – 7.29 (m, 3H, Ph), 6.53 (s,

1H, 4-H), 4.27 (q, J = 7.1 Hz, 2H, CH2CH3), 3.78 (d, J = 7.1 Hz, 2H, CH2-cy), 2.60 (s, 3H, CH3),

1.58 – 1.49 (m, 3H, cy), 1.41 – 1.31 (m, 6H, cy, CH2CH3), 1.06 – 0.95 (m, 3H, cy), 0.69 – 0.57 (m, 2H, cy).

13 C NMR (121 MHz, CDCl3) δ/ppm = 165.9 (COOEt), 137.0 (C-2), 134.1 (qPh), 133.7 (C-5),

129.6 (Ph), 128.5 (Ph), 127.4 (Ph), 112.0 (C-3), 110.0 (C-4), 59.4 (CH2CH3), 50.2 (CH2-cy),

39.0 (cy), 30.6 (cy), 26.2 (cy), 25.8 (cy), 14.7 (CH2CH3), 12.1 (CH3).

-1 IR (ATR) ṽmax/cm = 2975, 2926, 2850, 1738, 1698, 1420, 1242, 1224, 1191, 1062, 772, 702.

+ HRMS (ESI): calcd. for C21H28NO2 (M+H) 326.21146; found 326.21121.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

151 EXPERIMENTAL PART

1-(Cyclohexylmethyl)-2-methyl-5-phenyl-1H-pyrrole-3-carboxylic acid – SGA-50 (100)

According to general procedure D, LiOH (38.7 mg, 1.54 mmol, 10 eq.) and a solution of SGA-

43 (96, 50.0 mg, 0.154 mmol, 1.0 eq.) in dioxane/H2O (1.3 mL) were used. After 1 h the reaction was completed and gave SGA-50 (100) as colorless solid (40.4 mg, 0.136 mmol, 88%). The compound is literature known, but no analytical data are available[103].

Rf = 0.18 (6:1 hexanes/EtOAc). m.p.: 196 °C.

1 H NMR (500 MHz, CDCl3) δ/ppm = 7.42 – 7.38 (m, 2H, Ph), 7.36 – 7.32 (m, 3H, Ph), 6.59 (s,

1H, 4-H), 3.80 (d, J = 7.2 Hz, 2H, CH2), 2.62 (s, 3H, CH3), 1.59 – 1.52 (m, 3H, cy), 1.42 – 1.33 (m, 3H, cy), 1.06 – 0.97 (m, 3H, cy), 0.68 – 0.59 (m, 2H, cy).

13 C NMR (101 MHz, CDCl3) δ/ppm = 170.8 (COOH), 138.3 (C-2), 134.5 (C-5), 133.5 (qPh),

129.7 (Ph), 128.5 (Ph), 127.5 (Ph), 111.2 (C-3), 110.7 (C-4), 50.3 (CH2), 39.0 (cy), 30.6 (cy),

26.2 (cy), 25.8 (cy), 12.3 (CH3).

-1 IR (ATR) ṽmax/cm = 3030, 2971, 2921, 2848, 1738, 1660, 1533, 1435, 1364, 1267, 1227, 1205, 778, 768, 712, 703.

- HRMS (ESI): calcd. for C19H22NO2 (M-H) 296.16572; found 296.16560.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

Ethyl 1-(cyclohexylmethyl)-5-(2,5-dichlorophenyl)-2-methyl-1H-pyrrole-3-carboxylate – SGA-48 (110)

152 EXPERIMENTAL PART

Following general procedure C, ethyl acetoacetate (94, 208 µL, 1.65 mmol, 1.1 eq.) in dry THF (5.0 mL), NaH (90.0 mg, 2.25 mmol, 1.5 eq.) and a solution of 2-bromo-1-(2,5- dichlorophenyl)ethan-1-one (402 mg, 1.50 mmol, 1.0 eq.) in dry THF (1.0 mL) were used. Then, the residue was dissolved in acetic acid (5.0 mL) and cyclohexanemethanamine (95, 390 µL, 3.00 mmol, 2.0 eq.) was added. FCC (hexanes/EtOAc 9:1), followed by recrystallization from EtOH yielded SGA-48 (100) as colorless solid (271 mg, 0.686 mmol, 46%).

Rf = 0.48 (9:1 hexanes/EtOAc). m.p.: 98 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.48 (d, J = 2.0 Hz, 1H, 6’-H), 7.29 (dd, J = 8.2, 2.0 Hz, 1H, 4’-H), 7.25 (d, J = 9.1 Hz, 1H, 3’-H, collapses with chloroform), 6.51 (s, 1H, 4-H), 4.27 (q,

J = 7.0 Hz, 2H, CH2CH3), 3.55 (d, J = 7.0 Hz, 2H, CH2-cy), 2.59 (s, 3H, CH3), 1.61 – 1.56 (m,

3H, cy), 1.40 – 1.31 (m, 6H, cy, CH2CH3), 1.08 – 0.99 (m, 3H, cy), 0.68 – 0.58 (m, 2H, cy).

13 C NMR (101 MHz, CDCl3) δ/ppm = 165.6 (COOEt), 137.0 (C-2), 135.9 (C-1’ or C-5’), 134.8 (C-1’ or C-5’), 134.0 (C-3’), 131.2 (C-2’), 129.6 (C-6’), 129.2 (C-5), 127.2 (C-4’), 112.2 (C-3),

111.1 (C-4), 59.5 (CH2CH3), 50.8 (CH2-cy), 39.1 (cy), 30.6 (cy), 26.2 (cy), 25.8 (cy), 14.7

(CH2CH3), 12.1 (CH3).

-1 IR (ATR) ṽmax/cm = 2981, 2923, 2845, 1739, 1723, 1695, 1565, 1454, 1262, 1238, 1201, 1159, 1076, 1066, 800, 771.

35 + HRMS (ESI): calcd. for C21H26 Cl2NO2 (M+H) 394.13351; found 394.13343.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

1-(Cyclohexylmethyl)-5-(2,5-dichlorophenyl)-2-methyl-1H-pyrrole-3-carboxylic acid – SGA-52 (116)

According to general procedure D, LiOH (63.9 mg, 2.54 mmol, 10 eq.) and a solution of SGA-

48 (110, 100 mg, 0.254 mmol, 1.0 eq.) in dioxane/H2O (1.3 mL) were used. After 1 h the reaction was completed and gave SGA-52 (116) as colorless solid (75.7 mg, 0.207 mmol, 81%).

153 EXPERIMENTAL PART

Rf = 0.18 (6:1 hexanes/EtOAc). m.p.: 180 °C.

1 H NMR (500 MHz, CDCl3) δ/ppm = 7.48 (d, J = 2.0 Hz, 1H, 6’-H), 7.30 (dd, J = 8.2, 2.0 Hz, 1H, 4’-H), 7.26 (d, J = 8.2 Hz, 1H, 3’-H, collapses with chloroform), 6.57 (s, 1H, 4-H), 3.57 (d,

J = 6.4 Hz, 2H, CH2-cy), 2.60 (s, 3H, CH3), 1.63 – 1.57 (m, 3H, cy), 1.42 – 1.36 (m, 3H, cy), 1.08 – 1.00 (m, 3H, cy), 0.68 – 0.59 (m, 2H, cy).

13 C NMR (126 MHz, CDCl3) δ/ppm = 170.7 (COOH), 138.3 (C-2), 136.0 (C-1’ or C-5’), 135.0 (C-1’ or C-5’), 134.0 (C-3’), 131.0 (C-2’), 129.7 (C-6’), 129.6 (C-5), 127.2 (C-4’), 111.8 (C-4),

111.4 (C-3), 50.9 (CH2-cy), 39.0 (cy), 30.7 (cy), 26.2 (cy), 25.8 (cy), 12.3 (CH3).

-1 IR (ATR) ṽmax/cm = 3014, 2970, 2926, 2851, 1739, 1659, 1449, 1365, 1270, 1228, 1217, 1204, 814, 776.

35 - HRMS (ESI): calcd. for C19H20 Cl2NO2 (M-H) 364.08766; found 364.08783.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

Ethyl 5-(5-bromo-2-methoxyphenyl)-1-(cyclohexylmethyl)-2-methyl-1H-pyrrole-3- carboxylate – SGA-54 (97)

Following general procedure C, ethyl acetoacetate (94, 143 µL, 1.10 mmol, 1.1 eq.) in dry THF (5.0 mL), NaH (60.0 mg, 1.50 mmol, 1.5 eq.) and a solution of 2-bromo-1-(5-bromo-2- methoxyphenyl)ethan-1-one (93, 308 mg, 1.00 mmol, 1.0 eq.) in dry THF (1.0 mL) were used. Then, the residue was dissolved in acetic acid (5.0 mL) and cyclohexanemethanamine (95, 260 µL, 2.00 mmol, 2.0 eq.) was added. FCC (hexanes/EtOAc 9:1) yielded SGA-54 (97) as colorless solid (411 mg, 0.947 mmol, 95%).

Rf = 0.37 (6:1 hexanes/EtOAc). m.p.: 83 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.45 (dd, J = 8.7, 2.5 Hz, 1H, 4’-H), 7.37 (d, J = 2.5 Hz,

1H, 6’-H), 6.81 (d, J = 8.7 Hz, 1H, 3’-H), 6.48 (s, 1H, 4-H), 4.25 (q, J = 7.1 Hz, 2H, CH2CH3),

154 EXPERIMENTAL PART

3.76 (s, 3H, OCH3), 3.56 (d, J = 7.2 Hz, 2H, CH2-cy), 2.58 (s, 3H, CH3), 1.60 – 1.54 (m, 3H, cy), 1.41 – 1.29 (m, 6H, cy, CH2CH3), 1.08 – 0.97 (m, 3H, cy), 0.68 – 0.57 (m, 2H, cy).

13 C NMR (101 MHz, CDCl3) δ/ppm = 165.7 (COOEt), 156.6 (C-2’), 136.9 (C-2), 135.2 (C-6’), 132.3 (C-4’), 128.9 (C-5), 124.8 (C-1’), 112.8 (C-5’), 112.6 (C-3’), 112.1 (C-3), 110.6 (C-4),

59.3 (CH2CH3), 55.9 (OCH3), 50.9 (CH2-cy), 39.0 (cy), 30.7 (cy), 26.3 (cy), 25.8 (cy), 14.7

(CH2CH3), 12.1 (CH3).

-1 IR (ATR) ṽmax/cm = 2979, 2928, 2849, 1695, 1676, 1473, 1461, 1434, 1253, 1234, 1187, 1176, 1060, 1048, 1027, 774, 619.

79 + HRMS (ESI): calcd. for C22H29 BrNO3 (M+H) 434.13253; found 434.13229.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

5-(5-Bromo-2-methoxyphenyl)-1-(cyclohexylmethyl)-2-methyl-1H-pyrrole-3-carboxylic acid – SGA-55 (115)

According to general procedure D, LiOH (88.4 mg, 3.51 mmol, 10 eq.) and a solution of SGA-

54 (97, 152 mg, 0.351 mmol, 1.0 eq.) in dioxane/H2O (1.3 mL) were used. After 1 h the reaction was completed and gave SGA-55 (115) as colorless solid (90.0 mg, 0.222 mmol, 63%).

Rf = 0.72 (1:1 hexanes/EtOAc). m.p.: 224 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.46 (dd, J = 8.8, 2.5 Hz, 1H, 4’-H), 7.38 (d, J = 2.5 Hz,

1H, 6’-H), 6.82 (d, J = 8.8 Hz, 1H, 3’-H), 6.54 (s, 1H, 4-H), 3.77 (s, 3H, OCH3), 3.57 (d, J = 7.2

Hz, 2H, CH2-cy), 2.59 (s, 3H, CH3), 1.62 – 1.52 (m, 3H, cy), 1.43 – 1.33 (m, 3H, cy), 1.10 – 0.98 (m, 3H, cy), 0.69 – 0.58 (m, 2H, cy).

13 C NMR (101 MHz, CDCl3) δ/ppm = 170.4 (COOH), 156.6 (C-2’), 138.2 (C-2), 135.2 (C-6’), 132.4 (C-4’), 129.3 (C-5), 124.6 (C-1’), 112.8 (C-5’), 112.6 (C-3’), 111.4 (C-4), 111.3 (C-3),

55.9 (OCH3), 51.0 (CH2-cy), 39.0 (cy), 30.7 (cy), 26.3 (cy), 25.8 (cy), 12.3 (CH3).

-1 IR (ATR) ṽmax/cm = 3027, 2969, 2926, 2850, 1739, 1658, 1476, 1462, 1442, 1362, 1274, 1244, 1205, 1018, 808, 782, 619.

155 EXPERIMENTAL PART

79 - HRMS (ESI): calcd. for C20H23 BrNO3 (M-H) 404.08668; found 404.08697.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

Ethyl 1-(cyclohexylmethyl)-5-(4-fluorophenyl)-2-methyl-1H-pyrrole-3-carboxylate – SGA-59 (111)

Following general procedure C, ethyl acetoacetate (94, 208 µL, 1.65 mmol, 1.1 eq.) in dry THF (5.0 mL), NaH (90.0 mg, 2.25 mmol, 1.5 eq.) and a solution of 2-chloro-1-(4- fluorophenyl)ethan-1-one (259 mg, 1.50 mmol, 1.0 eq.) and KI (249 mg, 1.50 mmol, 1.0 eq.) in dry THF (3.0 mL) were used. Then, the residue was dissolved in acetic acid (5.0 mL) and cyclohexanemethanamine (95, 390 µL, 3.00 mmol, 2.0 eq.) was added. FCC (hexanes/EtOAc 9:1) yielded SGA-59 (111) as yellow oil (499 mg, 1.45 mmol, 97%).

Rf = 0.43 (9:1 hexanes/EtOAc).

1 H NMR (500 MHz, CDCl3) δ/ppm = 7.28 – 7.23 (m, 2H, 2’-H, 6’-H), 7.10 – 7.01 (m, 2H, 3’-H,

5’-H), 6.48 (s, 1H, 4-H), 4.25 (q, J = 7.1 Hz, 2H, CH2CH3), 3.71 (d, J = 7.2 Hz, 2H, CH2-cy),

2.57 (s, 3H, CH3), 1.58 – 1.48 (m, 3H, cy), 1.38 – 1.28 (m, 6H, cy, CH2CH3), 1.06 – 0.89 (m, 3H, cy), 0.68 – 0.53 (m, 2H, cy).

13 C NMR (121 MHz, CDCl3) δ/ppm = 165.7 (COOH), 162.2 (d, JCF = 247.0 Hz, C-4’), 136.9 (C-

2), 132.8 (C-5), 131.3 (d, JCF = 8.0 Hz, C-2’, C-6’), 129.7 (d, JCF = 3.3 Hz, C-1’), 115.4 (d, JCF

= 21.3 Hz, C-3’, C-5’), 111.9 (C-3), 110.0 (C-4), 59.3 (CH2CH3), 50.1 (CH2-cy), 39.0 (cy), 30.5

(cy), 26.1 (cy), 25.7 (cy), 14.6 (CH2CH3), 12.0 (CH3).

-1 IR (ATR) ṽmax/cm = 2977, 2925, 2853, 1693, 1242, 1227, 1218, 1195, 1152, 1062, 844, 811, 774.

+ HRMS (ESI): calcd. for C21H27FNO2 (M+H) 344.20203; found 344.20193.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

156 EXPERIMENTAL PART

1-(Cyclohexylmethyl)-5-(4-fluorophenyl)-2-methyl-1H-pyrrole-3-carboxylic acid – SGA-66 (117)

According to general procedure D, LiOH (169 mg, 6.71 mmol, 10 eq.) and a solution of SGA-

59 (111, 230 mg, 0.671 mmol, 1.0 eq.) in dioxane/H2O (3.0 mL) were used. After 2 h the reaction was completed and gave SGA-66 (117) as colorless solid (186 mg, 0.590 mmol, 88%).

Rf = 0.24 (6:1 hexanes/EtOAc). m.p.: 171 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.35 – 7.27 (m, 2H, 2’-H, 6’-H), 7.14 – 7.04 (m, 2H, 3’-H,

5’-H), 6.57 (s, 1H, 4-H), 3.75 (d, J = 7.1 Hz, 2H, CH2-cy), 2.61 (s, 3H, CH3), 1.63 – 1.51 (m, 3H, cy), 1.41 – 1.32 (m, 3H, cy), 1.08 – 0.97 (m, 3H, cy), 0.70 – 0.57 (m, 2H, cy).

13 C NMR (121 MHz, CDCl3) δ/ppm = 171.2 (COOH), 162.37 (d, JCF = 247.1 Hz, C-4’), 138.3

(C-2), 133.3 (C-5), 131.42 (d, JCF = 8.1 Hz, C-2’, C-6’), 129.6 (d, JCF = 3.4 Hz, C-1’), 115.57 (d,

JCF = 21.5 Hz, C-3’, C-5’), 111.2 (C-3), 110.8 (C-4), 50.3 (CH2-cy), 39.0 (cy), 30.6 (cy), 26.2

(cy), 25.8 (cy), 12.3 (CH3).

-1 IR (ATR) ṽmax/cm = 2927, 2854, 1739, 1652, 1568, 1494, 1449, 1265, 1223, 1203, 1158, 840, 776, 582.

- HRMS (ESI): calcd. for C19H21FNO2 (M-H) 314.15618; found 314.15635.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

Ethyl 1-(cyclohexylmethyl)-5-(4-methoxyphenyl)-2-methyl-1H-pyrrole-3-carboxylate – SGA-61 (112)

Following general procedure C, ethyl acetoacetate (94, 208 µL, 1.65 mmol, 1.1 eq.) in dry THF (5.0 mL), NaH (90.0 mg, 2.25 mmol, 1.5 eq.) and a solution of 2-bromo-1-(4-

157 EXPERIMENTAL PART methoxyphenyl)ethan-1-one (344 mg, 1.50 mmol, 1.0 eq.) and KI (249 mg, 1.50 mmol, 1.0 eq.) in dry THF (3.0 mL) were used. Then, the residue was dissolved in acetic acid (5.0 mL) and cyclohexanemethanamine (95, 390 µL, 3.00 mmol, 2.0 eq.) was added. FCC (hexanes/EtOAc 9:1), followed by recrystallization from EtOH yielded SGA-61 (112) as colorless solid (274 mg, 0.770 mmol, 51%).

Rf = 0.37 (9:1 hexanes/EtOAc). m.p.: 88 °C.

1 H NMR (500 MHz, CDCl3) δ/ppm = 7.25 – 7.21 (m, 2H, 2’-H, 6’-H), 6.95 – 6.89 (m, 2H, 3’-H,

5’-H), 6.47 (s, 1H, 4-H), 4.27 (q, J = 7.1 Hz, 2H, CH2CH3), 3.84 (s, 3H, OCH3), 3.73 (d, J = 7.3

Hz, 2H, CH2-cy), 2.58 (s, 3H, CH3), 1.60 – 1.51 (m, 3H, cy), 1.42 – 1.31 (m, 6H, cy, CH2CH3), 1.07 – 0.96 (m, 3H, cy), 0.70 – 0.59 (m, 2H, cy).

13 C NMR (126 MHz, CDCl3) δ/ppm = 165.9 (COOH), 159.0 (C-4’), 136.6 (C-2), 133.8 (C-5),

130.9 (C-2’, C-6’), 126.1 (C-1’), 113.9 (C-3’, C-5’), 111.7 (C-3), 109.5 (C-4), 59.3 (CH2CH3),

55.4 (OCH3), 50.1 (CH2-cy), 39.0 (cy), 30.6 (cy), 26.2 (cy), 25.8 (cy), 14.7 (CH2CH3), 12.1

(CH3).

-1 IR (ATR) ṽmax/cm = 3016, 2970, 2928, 2847, 1739, 1693, 1568, 1532, 1496, 1443, 1424, 1373, 1243, 1226, 1195, 1175, 1064, 1031, 835, 817, 795, 774.

+ HRMS (ESI): calcd. for C22H30NO3 (M+H) 356.22202; found 356.22192.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

1-(Cyclohexylmethyl)-5-(4-methoxyphenyl)-2-methyl-1H-pyrrole-3-carboxylic acid – SGA-67 (118)

According to general procedure D, LiOH (78.7 mg, 3.12 mmol, 10 eq.) and a solution of SGA-

61 (112, 111 mg, 0.312 mmol, 1.0 eq.) in dioxane/H2O (1.3 mL) were used. After 2 h the reaction was completed and gave SGA-67 (118) as colorless solid (95.3 mg, 0.291 mmol, 90%).

Rf = 0.20 (6:1 hexanes/EtOAc).

158 EXPERIMENTAL PART m.p.: 198 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.27 – 7.23 (m, 2H, 2’-H, 6’-H), 6.95 – 6.91 (m, 2H, 3’-H,

5’-H), 6.54 (s, 1H, 4-H), 3.85 (s, 3H, OCH3), 3.75 (d, J = 7.2 Hz, 2H, CH2-cy), 2.60 (s, 3H, CH3), 1.62 – 1.53 (m, 3H, cy), 1.43 – 1.33 (m, 3H, cy), 1.09 – 0.98 (m, 3H, cy), 0.70 – 0.59 (m, 2H, cy).

13 C NMR (101 MHz, CDCl3) δ/ppm = 171.3 (COOH), 159.0 (C-4’), 137.8 (C-2), 134.1 (C-5),

131.0 (C-2’, C-6’), 125.9 (C-1’), 113.9 (C-3’, C-5’), 111.0 (C-3), 110.2 (C-4), 55.3 (OCH3), 50.2

(CH2-cy), 38.9 (cy), 30.5 (cy), 26.2 (cy), 25.8 (cy), 12.3 (CH3).

-1 IR (ATR) ṽmax/cm = 3027, 3002, 2970, 2925, 2849, 1738, 1652, 1569, 1535, 1494, 1435, 1364, 1266, 1247, 1228, 1203, 840, 778.

- HRMS (ESI): calcd. for C20H24NO3 (M-H) 326.17617; found 326.17633.

Purity (HPLC): 93% (λ = 210 nm), > 96% (λ = 254 nm).

Ethyl 1-(cyclohexylmethyl)-5-(2,4-difluorophenyl)-2-methyl-1H-pyrrole-3-carboxylate – SGA-62 (113)

Following general procedure C, ethyl acetoacetate (94, 208 µL, 1.65 mmol, 1.1 eq.) in dry THF (5.0 mL), NaH (90.0 mg, 2.25 mmol, 1.5 eq.) and a solution of 2-chloro-1-(2,4- difluorophenyl)ethan-1-one (286 mg, 1.50 mmol, 1.0 eq.) and KI (249 mg, 1.50 mmol, 1.0 eq.) in dry THF (3.0 mL) were used. Then, the residue was dissolved in acetic acid (5.0 mL) and cyclohexanemethanamine (95, 390 µL, 3.00 mmol, 2.0 eq.) was added. FCC (hexanes/EtOAc 9:1) yielded SGA-62 (113) as yellow solid (424 mg, 1.17 mmol, 78%).

Rf = 0.43 (9:1 hexanes/EtOAc). m.p.: 94 °C.

1 H NMR (500 MHz, CDCl3) δ/ppm = 7.27 (td, J = 8.4, 6.5 Hz, 1H, 6’-H), 6.96 – 6.85 (m, 2H, 3’-

H, 5’-H), 6.53 (s, 1H, 4-H), 4.26 (q, J = 7.1 Hz, 2H, CH2CH3), 3.60 (d, J = 7.1 Hz, 2H, CH2-cy),

2.58 (s, 3H, CH3), 1.59 – 1.53 (m, 3H, cy), 1.38 – 1.30 (m, 6H, cy, CH2CH3), 1.06 – 0.98 (m, 3H, cy), 0.68 – 0.58 (m, 2H, cy).

159 EXPERIMENTAL PART

13 C NMR (121 MHz, CDCl3) δ/ppm = 165.5 (COOEt), 162.9 (dd, JCF = 250.1, 11.6 Hz, C-4’),

160.2 (dd, JCF = 248.9, 12.0 Hz, C-2’), 137.2 (C-2), 133.5 (dd, JCF = 9.4, 4.1 Hz, C-6’), 126.2

(C-5), 117.6 (dd, JCF = 15.7, 3.9 Hz, C-1’), 112.3 (C-3), 111.6 (dd, JCF = 21.1, 3.7 Hz, C-5’),

111.3 (C-4), 104.20 (t, JCF = 25.8 Hz, C-3’), 59.4 (CH2CH3), 50.6 (d, JCF = 3.0 Hz, CH2-cy), 39.0

(cy), 30.5 (cy), 26.1 (cy), 25.7 (cy), 14.6 (CH2CH3), 12.0 (CH3).

-1 IR (ATR) ṽmax/cm = 2971, 2929, 2848, 1739, 1698, 1571, 1426, 1371, 1235, 1199, 1067, 834.

+ HRMS (ESI): calcd. for C21H26F2NO2 (M+H) 362.19261; found 362.19247.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

1-(Cyclohexylmethyl)-5-(2,4-difluorophenyl)-2-methyl-1H-pyrrole-3-carboxylic acid - SGA-68 (119)

According to general procedure D, LiOH (167 mg, 6.61 mmol, 10 eq.) and a solution of SGA-

62 (113, 239 mg, 0.661 mmol, 1.0 eq.) in dioxane/H2O (1.3 mL) were used. After 1 h the reaction was completed and gave SGA-68 (119) as colorless solid (170 mg, 0.511 mmol, 77%).

Rf = 0.29 (6:1 hexanes/EtOAc). m.p.: 168 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.33 – 7.25 (m, 1H, 6’-H, collapses with chloroform), 6.98

– 6.86 (m, 2H, 3’-H, 5’-H), 6.60 (s, 1H, 4-H), 3.62 (d, J = 7.1 Hz, 2H, CH2-cy), 2.61 (s, 3H, CH3), 1.65 – 1.51 (m, 3H, cy), 1.49 – 1.32 (m, 3H, cy), 1.14 – 0.94 (m, 3H, cy), 0.72 – 0.56 (m, 2H, cy).

13 C NMR (101 MHz, CDCl3) δ/ppm = 171.0 (COOH), 163.0 (dd, JCF = 250.3, 11.6 Hz, C-4’),

160.3 (dd, JCF = 249.0, 12.0 Hz, C-2’), 138.5 (C-2), 133.6 (dd, JCF = 9.5, 4.0 Hz, C-6’), 126.7

(C-5), 117.5 (dd, JCF = 15.8, 3.8 Hz, C-1’), 112.0 (C-3), 111.7 (dd, JCF = 21.3, 3.8 Hz, C-5’),

111.6 (C-4), 104.3 (t, JCF = 25.9 Hz, C-3’), 50.72 (d, JCF = 3.0 Hz, CH2-cy), 39.0 (cy), 30.6 (cy),

26.2 (cy), 25.7 (cy), 12.2 (CH3).

-1 IR (ATR) ṽmax/cm = 2970, 2926, 2854, 1739, 1666, 1573, 1450, 1433, 1364, 1265, 1239, 1200, 1140, 778.

160 EXPERIMENTAL PART

- HRMS (ESI): calcd. for C19H20F2NO2 (M-H) 332.14676; found 332.14697.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

5-(5-Bromo-2-(trifluoromethoxy)phenyl)-2-methyl-1-pentyl-1H-pyrrole-3-carboxylic acid – SGA-149 (123)

Following general procedure C, ethyl acetoacetate (94, 87.6 µL, 0.693 mmol, 1.1 eq.) in dry THF (4.0 mL), NaH (37.8 mg, 0.945 mmol, 1.5 eq.) and a solution of ketone 102 (200 mg, 0.630 mmol, 1.0 eq.) and KI (105 mg, 0.630 mmol, 1.0 eq.) in dry THF (2.0 mL) was used. Then, the residue was dissolved in acetic acid (5.0 mL) and n-pentylamine (146 µL, 1.26 mmol, 2.0 eq.) was added. FCC (hexanes/EtOAc 99:1) yielded ethyl 5-(5-bromo-2- (trifluoromethoxy)phenyl)-2-methyl-1-pentyl-1H-pyrrole-3-carboxylate as colorless oil (81.5 mg, 0.176 mmol). The product was used without further purification or characterization for the next step.

Rf = 0.51 (9:1 hexanes/EtOAc).

According to general procedure D, LiOH (44.4 mg, 1.76 mmol, 10 eq.) and a solution of this ester (81.5 mg, 0.176 mmol, 1.0 eq.) in dioxane/H2O (3.0 mL) were used. After 18 h the reaction was completed and gave SGA-149 (123) as colorless solid (40.5 mg, 0.0933 mmol, 15% over two steps).

Rf = 0.06 (9:1 hexanes/EtOAc). m.p.: 121 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.57 – 7.51 (m, 2H, 4’’-H, 6’’-H), 7.24 – 7.20 (m, 1H, 3’’-

H), 6.61 (s, 1H, 4-H), 3.74 – 3.68 (m, 2H, 1’-H), 2.61 (s, 3H, CH3), 1.49 – 1.43 (m, 2H, 2’-H), 1.19 – 1.06 (m, 4H, 3’-H, 4’-H), 0.80 (t, J = 7.2 Hz, 3H, 5’-H).

13 C NMR (101 MHz, CDCl3) δ/ppm = 169.4 (COOH), 146.6 (C-2’’), 138.3 (C-2), 135.9 (C-6’’),

132.8 (C-4’’), 128.7 (C-5’’), 126.1 (C-5), 122.5 (q, J = 279.8 Hz, OCF3), 122.4 (C-3’’), 120.0 (C- 1’’), 112.7 (C-4), 111.4 (C-3), 44.7 (C-1’), 30.2 (C-2’), 28.8 (C-3’), 22.1 (C-4’), 13.9 (C-5’), 11.9

(CH3).

161 EXPERIMENTAL PART

-1 IR (ATR) ṽmax/cm = 2929, 1663, 1471, 1436, 1247, 1212, 1194, 1160, 781.

79 + HRMS (ESI): calcd. for C18H20 BrF3NO3 (M+H) 434.05732; found 434.05765.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

1-Benzyl-5-(5-bromo-2-(trifluoromethoxy)phenyl)-2-methyl-1H-pyrrole-3-carboxylic acid – SGA-150 (125)

Following general procedure C, ethyl acetoacetate (94, 104 µL, 0.821 mmol, 1.1 eq.) in dry THF (4.0 mL), NaH (44.8 mg, 1.12 mmol, 1.5 eq.) and a solution of ketone 102 (237 mg, 0.746 mmol, 1.0 eq.) and KI (124 mg, 0.746 mmol, 1.0 eq.) in dry THF (2.0 mL) were used. Then, the residue was dissolved in acetic acid (5.0 mL) and benzylamine (204 µL, 1.87 mmol, 2.5 eq.) was added. FCC (hexanes/EtOAc 97:3) yielded ethyl 5-(5-bromo-2- (trifluoromethoxy)phenyl)-1-isopropyl-2-methyl-1H-pyrrole-3-carboxylate as colorless oil (107 mg, 0.222 mmol). The product was used without further purification or characterization for the next step.

Rf = 0.44 (9:1 hexanes/EtOAc).

According to general procedure D, LiOH (55.9 mg, 2.22 mmol, 10 eq.) and a solution of this ester (107 mg, 0.222 mmol, 1.0 eq.) in dioxane/H2O (3.0 mL) were used. After 16 h the reaction was completed and gave SGA-150 (125) as colorless solid (34.4 mg, 0.0757 mmol, 10% over two steps).

Rf = 0.27 (9:1 hexanes/EtOAc). m.p.: 185 °C.

1 H NMR (400 MHz, CD2Cl2) δ/ppm = 11.12 (s, 1H, COOH), 7.51 (dd, J = 8.8, 2.5 Hz, 1H, 4’- H), 7.39 (d, J = 2.5 Hz, 1H, 6’-H), 7.30 – 7.18 (m, 4H, 3’-H, Ph), 6.83 – 6.77 (m, 2H, Ph), 6.70

(s, 1H, 4-H), 5.00 (s, 2H, CH2), 2.49 (s, 3H, CH3).

13 C NMR (121 MHz, CD2Cl2) δ/ppm = 169.9 (COOH), 147.0 (C-2’), 139.6 (C-2), 137.4 (C-1’), 136.3 (C-6’), 133.4 (C-4’), 129.3 (Ph), 128.5 (qPh), 128.0 (Ph), 127.3 (C-5), 126.2 (Ph), 122.8

162 EXPERIMENTAL PART

(C-3’), 120.8 (q, JCF = 258.7 Hz, OCF3), 120.3 (C-5’), 113.1 (C-4), 112.3 (C-3), 48.7 (CH2), 12.2

(CH3).

-1 IR (ATR) ṽmax/cm = 2925, 2360, 1670, 1249, 1223, 1198, 1171, 733.

79 - HRMS (ESI): calcd. for C20H14 BrF3NO3 (M-H) 452.01146; found 452.01168.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

5-(5-Bromo-2-(trifluoromethoxy)phenyl)-1-(cyclohexylmethyl)-2-ethyl-1H-pyrrole-3- carboxylic acid – SGA-152 (121)

Following general procedure C, ethyl propionylacetate (99.9 µL, 0.693 mmol, 1.1 eq.) in dry THF (4.0 mL), NaH (37.8 mg, 0.945 mmol, 1.5 eq.) and a solution of ketone 102 (200 mg, 0.630 mmol, 1.0 eq.) and KI (105 mg, 0.630 mmol, 1.0 eq.) in dry THF (2.0 mL) were used. Then, the residue was dissolved in acetic acid (5.0 mL) and cyclohexanemethanamine (95, 164 µL, 1.26 mmol, 2.0 eq.) was added. FCC (hexanes/EtOAc 99:1) yielded ethyl 5-(5-bromo- 2-(trifluoromethoxy)phenyl)-1-(cyclohexylmethyl)-2-ethyl-1H-pyrrole-3-carboxylate as colorless oil (98.0 mg, 0.195 mmol). The product was used without further purification or characterization for the next step.

Rf = 0.58 (9:1 hexanes/EtOAc).

According to general procedure D, LiOH (49.2 mg, 1.95 mmol, 10 eq.) and a solution of this ester (98.0 mg, 0.195 mmol, 1.0 eq.) in dioxane/H2O (3.0 mL) were used. After 18 h the reaction was completed and gave SGA-152 (121) as yellow solid (30.8 mg, 0.0649 mmol, 9% over two steps).

Rf = 0.24 (9:1 hexanes/EtOAc). m.p.: 152 °C.

1 H NMR (500 MHz, CDCl3) δ/ppm = 11.35 (s, 1H, COOH), 7.57 – 7.50 (m, 2H, 4’-H, 6’-H), 7.20

(dd, J = 8.6, 1.4 Hz, 1H, 3’-H), 6.61 (s, 1H, 4-H), 3.61 (d, J = 7.2 Hz, 2H, CH2-cy), 3.04 (q, J =

7.4 Hz, 2H, CH2CH3), 1.64 – 1.54 (m, 3H, cy), 1.39 – 1.31 (m, 3H, cy), 1.23 (t, J = 7.4 Hz, 3H,

CH2CH3), 1.08 – 0.99 (m, 3H, cy), 0.70 – 0.60 (m, 2H, cy).

163 EXPERIMENTAL PART

13 C NMR (121 MHz, CDCl3) δ/ppm = 169.8 (COOH), 146.3 (C-2’), 145.1 (C-2), 135.5 (C-4’ or

C-6’), 132.5 (C-4’ or C-6’), 129.1 (C-1’), 126.7 (C-5), 122.3 (C-3’), 120.3 (q, JCF = 259.2 Hz,

OCF3), 120.0 (C-5’), 113.2 (C-4), 110.8 (C-3), 50.9 (CH2-cy), 39.6 (cy), 30.7 (cy), 26.2 (cy),

25.8 (cy), 19.2 (CH2CH3), 14.5 (CH2CH3).

-1 IR (ATR) ṽmax/cm = 2927, 2359, 1659, 1469, 1441, 1250, 1212, 1192, 1172.

79 - HRMS (ESI): calcd. for C21H22 BrF3NO3 (M-H) 472.07406; found 472.07418.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

5-(5-Bromo-2-(trifluoromethoxy)phenyl)-1-isopropyl-2-methyl-1H-pyrrole-3-carboxylic acid – SGA-153 (124)

Following general procedure C, ethyl acetoacetate (94, 87.6 µL, 0.693 mmol, 1.1 eq.) in dry THF (4.0 mL), NaH (37.8 mg, 0.945 mmol, 1.5 eq.) and a solution of ketone 102 (200 mg, 0.630 mmol, 1.0 eq.) and KI (105 mg, 0.630 mmol, 1.0 eq.) in dry THF (2.0 mL) were used. Then, the residue was dissolved in acetic acid (5.0 mL) and isopropylamine (108 µL, 1.26 mmol, 2.0 eq.) was added. FCC (hexanes/EtOAc 99:1) yielded ethyl 5-(5-bromo-2- (trifluoromethoxy)phenyl)-1-isopropyl-2-methyl-1H-pyrrole-3-carboxylate as colorless oil (65.9 mg, 0.152 mmol). The product was used without further purification or characterization for the next step.

Rf = 0.55 (9:1 hexanes/EtOAc).

According to general procedure D, LiOH (38.3 mg, 1.52 mmol, 10 eq.) and a solution of this ester (65.9 mg, 0.152 mmol, 1.0 eq.) in dioxane/H2O (3.0 mL) were used. After 18 h the reaction was completed and gave SGA-153 (124) as colorless solid (32.4 mg, 0.0798 mmol, 12% over two steps).

Rf = 0.11 (9:1 hexanes/EtOAc). m.p.: 192 °C.

1 H NMR (400 MHz, C3D6O) δ/ppm = 7.75 (dd, J = 8.8, 2.6 Hz, 1H, 4’-H), 7.65 (d, J = 2.6 Hz, 1H, 6’-H), 7.44 (dq, J = 8.8, 1.5 Hz, 1H, 3’-H), 6.47 (s, 1H, 4-H), 4.27 (p, J = 7.0 Hz, 1H, CH),

2.72 (s, 3H, CH3), 1.45 (d, J = 7.0 Hz, 6H, CH(CH3)2).

164 EXPERIMENTAL PART

13 C NMR (101 MHz, C3D6O) δ/ppm = 166.4 (COOH), 147.7 (C-2’), 137.5 (C-2), 137.0 (C-6’),

134.0 (C-4’), 130.5 (C-1’), 126.4 (C-5), 123.7 (q, J = 257.6 Hz, OCF3), 123.5 (C-3’), 120.6 (C-

5’), 113.9 (C-3), 113.0 (C-4), 50.2 (CH), 22.0 (CH(CH3)2), 13.0 (CH3).

-1 IR (ATR) ṽmax/cm = 2936, 2358, 1672, 1248, 1214, 1200, 1166.

79 - HRMS (ESI): calcd. for C16H14 BrF3NO3 (M-H) 404.01146; found 404.01164.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

5-(5-Bromo-2-(trifluoromethoxy)phenyl)-1-(cyclohexylmethyl)-2-phenyl-1H-pyrrole-3- carboxylic acid – SGA-154 (122)

Following general procedure C, ethyl benzoylacetate (134 µL, 0.693 mmol, 1.1 eq.) in dry THF (4.0 mL), NaH (37.8 mg, 0.945 mmol, 1.5 eq.) and a solution of ketone 102 (200 mg, 0.630 mmol, 1.0 eq.) and KI (105 mg, 0.630 mmol, 1.0 eq.) in dry THF (2.0 mL) were used. Then, the residue was dissolved in acetic acid (5.0 mL) and cyclohexanemethanamine (95, 328 µL, 2.52 mmol, 4.0 eq.) was added. FCC (hexanes/EtOAc 99:1) yielded ethyl 5-(5-bromo-2- (trifluoromethoxy)phenyl)-1-(cyclohexylmethyl)-2-phenyl-1H-pyrrole-3-carboxylate as yellow solid (151 mg, 0.274 mmol). The product was used without further purification or characterization for the next step.

Rf = 0.50 (9:1 hexanes/EtOAc).

According to general procedure D, LiOH (69.2 mg, 2.74 mmol, 10 eq.) and a solution of this ester (151 mg, 0.274 mmol, 1.0 eq.) in dioxane/H2O (3.0 mL) were used. After 18 h the reaction was completed and gave SGA-154 (122) as yellow solid (91.5 mg, 0.175 mmol, 28% over two steps).

Rf = 0.12 (9:1 hexanes/EtOAc). m.p.: 170 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.61 (d, J = 2.5 Hz, 1H, 6’-H), 7.55 (dd, J = 8.7, 2.5 Hz, 1H, 4’-H), 7.47 – 7.41 (m, 3H, Ph), 7.39 – 7.35 (m, 2H, Ph), 7.23 (dq, J = 8.7, 1.3 Hz, 1H, 3’-

165 EXPERIMENTAL PART

H), 6.74 (s, 1H, 4-H), 3.56 (d, J = 7.4 Hz, 2H, CH2-cy), 1.48 – 1.42 (m, 3H, cy), 1.11 – 1.05 (m, 2H, cy), 1.03 – 0.95 (m, 1H, cy), 0.90 – 0.82 (m, 3H, cy), 0.43 – 0.32 (m, 2H, cy).

13 C NMR (101 MHz, CDCl3) δ/ppm = 167.7 (COOH), 146.1 (C-2’), 141.7 (C-2), 135.2 (C-6’), 132.7 (C-4’), 131.7 (C-5, C-1’, C-5’ or qPh), 131.0 (Ph), 128.8 (C-5, C-1’, C-5’ or qPh), 128.7

(Ph), 128.3 (Ph), 122.4 (C-3’), 120.4 (q, JCF = 266.3 Hz, OCF3), 120.2 (C-5, C-1’, C-5’ or qPh),

113.8 (C-4), 112.7 (C-3), 52.0 (CH2-cy), 38.7 (cy), 30.3 (cy), 26.1 (cy), 25.6 (cy). One quaternary carbon is missing.

-1 IR (ATR) ṽmax/cm = 2915, 2335, 1667, 1487, 1248, 1208, 1169, 1127, 796, 697.

79 - HRMS (ESI): calcd. for C25H22 BrF3NO3 (M-H) 520.07406; found 520.07418.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

Ethyl 1-(cyclohexylmethyl)-2-methyl-5-(2-(trifluoromethoxy)phenyl)-1H-pyrrole-3- carboxylate (114)

Following general procedure C, ethyl acetoacetate (94, 123 µL, 0.972 mmol, 1.1 eq.) in dry THF (4.0 mL), NaH (53.0 mg, 1.32 mmol, 1.5 eq.) and a solution of ketone 101 (250 mg, 0.883 mmol, 1.0 eq.) and KI (147 mg, 0.883 mmol, 1.0 eq.) in dry THF (2.0 mL) were used. Then, the residue was dissolved in acetic acid (5.0 mL) and cyclohexanemethanamine (95, 230 µL, 1.77 mmol, 2.0 eq.) was added. FCC (hexanes/EtOAc 9:1) yielded ester 114 as colorless solid (345 mg, 0.845 mmol, 95%).

Rf = 0.46 (9:1 hexanes/EtOAc). m.p.: 66 °C.

1 H NMR (100 MHz, CDCl3) δ/ppm = 7.44 – 7.29 (m, 4H, 3’-H, 4’-H, 5’-H, 6’-H), 6.53 (s, 1H, 4-

H), 4.27 (q, J = 7.1 Hz, 2H, CH2CH3), 3.59 (d, J = 7.1 Hz, 2H, CH2-cy), 2.59 (s, 3H, CH3), 1.59

– 1.52 (m, 3H, cy), 1.40 – 1.32 (m, 6H, cy, CH2CH3), 1.06 – 0.94 (m, 3H, cy), 0.67 – 0.54 (m, 2H, cy).

13 C NMR (101 MHz, CDCl3) δ/ppm = 165.8 (COOEt), 147.4 (C-2’), 137.1 (C-2), 133.4 (C-4’, C-5’ or C-6’), 129.6 (C-4’, C-5’ or C-6’), 127.9 (C-5), 126.9 (C-1’), 126.7 (C-4’, C-5’ or C-6’),

166 EXPERIMENTAL PART

120.5 (q, JCF = 257.4 Hz, OCF3), 120.4 (C-3’), 112.2 (C-3), 111.4 (C-4), 59.5 (CH2CH3), 50.7

(CH2-cy), 39.0 (cy), 30.6 (cy), 26.2 (cy), 25.8 (cy), 14.7 (CH2CH3), 12.1 (CH3).

-1 IR (ATR) ṽmax/cm = 1934, 1692, 1447, 1422, 1242, 1192, 1155, 1059, 769.

+ HRMS (ESI): calcd. for C22H27F3NO3 (M+H) 410.19375; found 410.19336.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

1-(Cyclohexylmethyl)-2-methyl-5-(2-(trifluoromethoxy)phenyl)-1H-pyrrole-3-carboxylic acid – SGA-162 (120)

According to general procedure D, LiOH (142 mg, 5.62 mmol, 10 eq.) and a solution of ester

114 (230 mg, 0.562 mmol, 1.0 eq.) in dioxane/H2O (3.0 mL) were used. After 2 h the reaction was completed and gave SGA-162 (120) as colorless solid (199 mg, 0.523 mmol, 93%).

Rf = 0.11 (9:1 hexanes/EtOAc). m.p.: 170 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 11.13 (s, 1H, COOH), 7.46 – 7.30 (m, 4H, 3’-H, 4’-H, 5’-

H, 6’-H), 6.59 (s, 1H, 4-H), 3.60 (d, J = 7.1 Hz, 2H, CH2-cy), 2.61 (s, 3H, CH3), 1.61 – 1.52 (m, 3H, cy), 1.41 – 1.31 (m, 3H, cy), 1.09 – 0.96 (m, 3H, cy), 0.67 – 0.55 (m, 2H, cy).

13 C NMR (101 MHz, CDCl3) δ/ppm = 169.9 (COOH), 147.4 (C-2’), 138.3 (C-2), 133.4 (C-4’, C-5’ or C-6’), 129.8 (C-4’, C-5’ or C-6’), 128.3 (C-5), 126.8 (C-4’, C-5’ or C-6’), 126.7 (C-1’),

120.5 (q, JCF = 260.2 Hz, OCF3), 120.4 (C-4’), 112.1 (C-4), 111.2 (C-3), 50.8 (CH2-cy), 38.9

(cy), 30.6 (cy), 26.2 (cy), 25.7 (cy), 12.3 (CH3).

-1 IR (ATR) ṽmax/cm = 2927, 1642, 1473, 1444, 1249, 1199, 1156, 776, 759.

- HRMS (ESI): calcd. for C20H21F3NO3 (M-H) 380.14790; found 380.14805.

Purity (HPLC): > 96% (λ = 210 nm).

167 EXPERIMENTAL PART

5.2 Preparation of TPC2 inhibitors 5.2.1 General procedures

General Procedure E – Protection

Following a general procedure published by Schrittwieser et al.[149], the appropriate amines or phenols (1.0 eq.) were dispersed in dry CH2Cl2 and cooled to 0 °C, followed by dropwise addition of NEt3 (1.5-3.5 eq.) and ethyl chloroformate (162, 1.3-2.5 eq.). The reaction mixture was stirred at rt or was refluxed for 4-72 h, before water was added. The mixture was extracted thrice with CH2Cl2, the combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification was accomplished by flash column chromatography (FCC) to yield carbamates and carbonates.

General Procedure F – Wittig reaction

(Methoxymethyl)-triphenylphosphonium chloride (1.2 eq.) was suspended in dry THF and cooled to -4 °C, before LDA (20% in THF/ethylbenzene/heptane, 1.0-1.2 eq.) was added dropwise. The mixture was stirred for 20 min. at -4 °C, followed by slow, dropwise addition of a solution of the appropriate aldehyde (1.0 eq.) in THF. The solution was stirred for 16-50 h, then poured on water and the mixture was extracted thrice with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The crude product was purified by FCC to give the desired cis/trans-enol ethers. The products were directly used for the next step without further analysis.

General Procedure G – N-acyl Pictet-Spengler reaction

Carbamate (1.0 eq.) and enol ether (1.0-1.2 eq.) were dissolved in dry CH2Cl2, 4 Å molecular sieves (1.00 g per 20 mL solvent) was added and the mixture was cooled to 0 °C, before TFA (10-11 eq.) was added dropwise. The mixture was allowed to warm to rt and stirred for 18-90 h, before the molecular sieves was removed by filtration and sat. aq. NaHCO3 solution was added. The mixture was extracted thrice with CH2Cl2, the combined organic layers were washed with 2 M aq. HCl solution and brine, dried over Na2SO4, filtered and concentrated in vacuo. Purification was accomplished by FCC to yield the desired racemic tetrahydroisoquinolines.

168 EXPERIMENTAL PART

General Procedure H – Lithium alanate reductions of nitroolefins, carbamates and carbonates

[152] According to Cava et al. , LiAlH4 (7-12 eq.) was suspended in dry THF and a solution of carbamate (1.0 eq.) in dry THF was added dropwise and the suspension was refluxed for 3-

20 h. The mixture was allowed to cool to rt and Na2SO4 x 10 H2O was added in small portions under vigorous stirring over 20 minutes. Water was added and the mixture was extracted thrice with EtOAc. The combined organic layers were extracted with 2 M aq. HCl solution, the acidic phase was neutralized with sat. aq. NaHCO3 solution and extracted thrice with EtOAc again.

This organic layer was dried over Na2SO4 and concentrated in vacuo to give tertiary amines if not otherwise described.

General Procedure I – Chan-Evans-Lam coupling

[155] According to Evans et al. , phenols (1.0 eq), Cu(OAc)2 (1.1 eq. per phenolic group), boronic acid (3.0 eq. per phenolic group) and 4 Å molecular sieves (1.00 g per 40 mL solvent) were suspended in dry CH2Cl2 and a mixture of NEt3 and pyridine (1:1, 2.5 eq. each) was added dropwise. The reaction mixture was stirred for 13-22 h at rt under ambient atmosphere, followed by filtration through a celite pad. The organic filtrate was washed with sat. aq. NaHCO3 solution, 10% aq. citric acid solution and brine, dried over Na2SO4, filtered and concentrated in vacuo. The crude products were purified by FCC or HPLC to obtain the desired diaryl ethers.

General Procedure J – Formation of hydrochloride-salts

To receive solid and stable compounds for biological experiments, the tertiary amines were dissolved in 1.25 M methanolic HCl solution (3.0 mL per 100 mg amine) and concentrated in vacuo to yield the amorphous hydrochloride salts as solids. Melting points were obtained from the hydrochloride salts, remaining analytical data from the free amines, unless stated otherwise.

169 EXPERIMENTAL PART

5.2.2 Synthetic procedures 5.2.2.1 Synthesis of arylethylamine building blocks

(E)-1-(Benzyloxy)-2-methoxy-4-(2-nitrovinyl)benzene (173)

Following a procedure published by Pouysegu et al.[148], 4-benzyloxy-3-methoxybenzaldehyde (172, 10.0 g, 41.3 mmol, 1.0 eq.) and ammonium acetate (2.80 g, 36.4 mmol, 0.90 eq.) were dissolved in glacial acetic acid (24 mL) and nitromethane (16.0 mL, 299 mmol, 7.4 eq.) was added under nitrogen atmosphere. The mixture was heated to reflux for 3 h. Water (5.0 mL) was added, the precipitate was collected by filtration and was washed with cold MeOH. The crude product was recrystallized from MeOH to yield nitroolefin 173 (7.08 g, 24.8 mmol, 62%) as yellow crystals. Analytical data are in accordance with literature.

Rf = 0.46 (4:1 hexanes/acetone). m.p.: 123 °C. [lit.[148]: 124-125 °C]

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.95 (d, J = 13.6 Hz, 1H, CHCHNO2), 7.51 (d, J = 13.6

Hz, 1H, CHCHNO2), 7.44 – 7.32 (m, 5H, PhCH2), 7.10 (dd, J = 8.4, 2.0 Hz, 1H, 5-H), 7.02 (d,

J = 2.0 Hz, 1H, 3-H), 6.92 (d, J = 8.4 Hz, 1H, 6-H), 5.22 (s, 2H, PhCH2), 3.93 (s, 3H, OCH3).

13 C NMR (101 MHz, CDCl3) δ/ppm = 152.1 (C-1), 150.2 (C-2), 139.4 (CHCHNO2), 136.2 (qPh),

135.4 (CHCHNO2), 128.9 (Ph), 128.4 (Ph), 127.4 (Ph), 124.5 (C-5), 123.2 (C-4), 113.6 (C-3),

111.0 (C-6), 71.0 (PhCH2), 56.3 (OCH3).

-1 IR (ATR) ṽmax/cm = 3112, 3048, 2980, 2358, 1629, 1491, 1258, 1224, 1142, 976, 805, 746, 697.

•+ HRMS (EI): calcd. for C16H15NO4 (M) 285.0996; found 285.0994.

Purity (HPLC): > 96% (λ = 210 nm).

170 EXPERIMENTAL PART

2-(4-(Benzyloxy)-3-methoxyphenyl)ethan-1-amine (171)

Similar to general procedure H, LiAlH4 (1.70 g, 44.8 mmol, 5.1 eq.) in dry THF (10 mL) and nitrovinyl (173, 2.53 g, 8.87 mmol, 1.0 eq.) in dry THF (20 mL) were used and the resulting mixture was stirred at 80 °C for 18 h. Amine 171 (1.44 g, 5.60 mmol, 63%) was isolated as yellow oil. Analytical data are in accordance with literature[148].

Rf = 0.30 (9:1 CH2Cl2/MeOH).

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.46 – 7.41 (m, 2H, 2’-H, 6’-H), 7.39 – 7.34 (m, 2H, 3’-H, 5’-H), 7.29 (ddd, J = 7.2, 4.8, 1.4 Hz, 1H, 4’-H), 6.82 (d, J = 8.1 Hz, 1H, 5-H), 6.75 (d, J = 2.0

Hz, 1H, 2-H), 6.67 (dd, J = 8.1, 2.0 Hz, 1H, 6-H), 5.13 (s, 2H, PhCH2), 3.88 (s, 3H, OCH3), 2.94

(t, J = 6.8 Hz, 2H, CH2CH2NH2), 2.68 (t, J = 6.8 Hz, 2H, CH2CH2NH2).

13 C NMR (101 MHz, CDCl3) δ/ppm = 149.7 (C-3), 146.7 (C-4), 137.4 (C-1’), 133.0 (C-1), 128.5 (C-3’, C-5’), 127.8 (C-4’), 127.3 (C-2’, C-6’), 120.8 (C-6), 114.3 (C-5), 112.7 (C-2), 71.2

(PhCH2), 56.0 (OCH3), 43.6 (CH2CH2NH2), 39.5 (CH2CH2NH2).

-1 IR (ATR) ṽmax/cm = 3032, 2938, 2909, 2869, 2838, 1512, 1260, 1227, 1139, 1032, 738, 696.

+ HRMS (ESI): calcd. for C16H20NO2 (M+H) 258.14886; found 258.14886.

Ethyl (4-(benzyloxy)-3-methoxyphenethyl)carbamate (170)

Following general procedure E, amine 171 (1.40 g, 5.44 mmol, 1.0 eq.) in CH2Cl2 (20 mL),

NEt3 (1.10 mL, 8.16 mmol, 1.5 eq.) and ethyl chloroformate (162, 680 µL, 7.07 mmol, 1.3 eq.) were used. The reaction was completed after 72 h and the product was purified by FCC (4:1 hexanes/acetone) to yield carbamate 170 (861 mg, 2.61 mmol, 48%) as light yellow solid. Analytical data are in accordance with literature[149].

Rf = 0.28 (4:1 hexanes/acetone).

171 EXPERIMENTAL PART m.p.: 76 °C. [lit.[149]: 80-81 °C]

1 H NMR (500 MHz, CDCl3) δ/ppm = 7.43 (dt, J = 8.1, 1.9 Hz, 2H, 2’-H, 6’-H), 7.36 (ddt, J = 8.1, 6.4, 1.3 Hz, 2H, 3’-H, 5’-H), 7.32 – 7.28 (m, 1H, 4’-H), 6.82 (d, J = 8.1 Hz, 1H, 5-H), 6.76 – 6.70

(m, 1H, 2-H), 6.66 (dd, J = 8.1, 2.0 Hz, 1H, 6-H), 5.13 (s, 2H, PhCH2), 4.65 (s, 1H, NH), 4.11

(q, J = 7.0 Hz, 2H, CH2CH3), 3.88 (s, 3H, OCH3), 3.40 (q, J = 6.8 Hz, 2H, CH2CH2NH), 2.74 (t,

J = 6.8 Hz, 2H, CH2CH2NH), 1.23 (t, J = 7.0 Hz, 3H, CH2CH3).

13 C NMR (126 MHz, CDCl3) δ/ppm = 156.7 (CO), 149.9 (C-3), 147.0 (C-4), 137.4 (C-1’), 132.1 (C-1), 128.7 (C-3’, C-5’), 127.9 (C-4’), 127.4 (C-2’, C-6’), 120.8 (C-6), 114.4 (C-5), 112.6 (C-

2), 71.3 (PhCH2), 60.9 (CH2CH3), 56.1 (OCH3), 42.3 (CH2CH2NH), 35.9 (CH2CH2NH), 14.8

(CH2CH3).

-1 IR (ATR) ṽmax/cm = 3333, 2980, 2936, 1682, 1515, 1252, 1228, 1138.

+ HRMS (ESI): calcd. for C19H24NO4 (M+H) 330.16998; found 330.17056.

Purity (HPLC): > 96% (λ = 210 nm).

Ethyl (4-((ethoxycarbonyl)oxy)-3-methoxyphenethyl)carbamate (165)

Following general procedure E, 3-methoxytyramine hydrochloride (160, 5.00 g, 24.5 mmol, 1.0 eq.) in CH2Cl2 (25 mL), NEt3 (12.0 mL, 58.9 mmol, 3.5 eq.) and ethyl chloroformate (162, 5.90 mL, 61.4 mmol, 2.5 eq.) were used. The reaction was completed after 16 h and the product was purified by FCC (4:1 hexanes/acetone) to yield carbamate 165 (5.86 g, 18.8 mmol, 77%) as colorless solid.

Rf = 0.10 (CH2Cl2). m.p.: 68 °C.

1 H NMR (500 MHz, CDCl3) δ/ppm = 7.05 (d, J = 8.0 Hz, 1H, 5-H), 6.81 – 6.74 (m, 2H, 2-H, 6-

H), 4.68 (s, 1H, NH), 4.31 (q, J = 7.0 Hz, 2H, OCOOCH2CH3), 4.11 (q, J = 7.0 Hz, 2H,

NCOOCH2CH3), 3.84 (s, 3H, OCH3), 3.43 (q, J = 6.5 Hz, 2H, CH2CH2NH), 2.80 (t, J = 6.5 Hz,

2H, CH2CH2NH), 1.38 (t, J = 7.0 Hz, 3H, OCOOCH2CH3), 1.23 (t, J = 7.0 Hz, 3H,

NCOOCH2CH3).

172 EXPERIMENTAL PART

13 C NMR (126 MHz, CDCl3) δ/ppm = 156.7 (N-CO), 153.5 (O-CO), 151.3 (C-3), 138.9 (C-4),

138.1 (C-1), 122.5 (C-5), 120.9 (C-6), 113.2 (C-2), 65.1 (OCOOCH2CH3), 60.9

(NCOOCH2CH3), 56.1 (OCH3), 42.1 (CH2CH2NH), 36.3 (CH2CH2NH), 14.8 (OCOOCH2CH3),

14.3 (NCOOCH2CH3).

-1 IR (ATR) ṽmax/cm = 3336, 2980, 2940, 2872, 1750, 1686, 1541, 1514, 1283, 1251, 1208, 1059, 1032.

+ HRMS (ESI): calcd. for C15H22NO6 (M+H) 312.14416; found 312.14425.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

Ethyl (4-hydroxy-3-methoxyphenethyl)carbamate (166)

Following general procedure E, 3-methoxytyramine hydrochloride (160, 10.2 g, 50.0 mmol, 1.0 eq.) in CH2Cl2 (50 mL), NEt3 (24.0 mL, 175 mmol, 3.5 eq.) and ethyl chloroformate (162, 12.0 mL, 125 mmol, 2.5 eq.) were used. After 18 h, a 1 M ethanolic NaOH solution (100 mL) was added and the mixture was stirred for 3.5 h. The reaction mixture was neutralized using

2 M aq. HCl solution, followed by extraction with CH2Cl2 (4 x 80 mL). The combined organic layers were dried over Na2SO4 and concentrated in vacuo. Purification was accomplished by FCC (4:1 hexanes/acetone) to yield carbamate 166 (3.49 g, 14.6 mmol, 29%) as colorless solid. Analytical data are in accordance with literature[73].

Rf = 0.18 (4:1 hexanes/acetone). m.p.: 98 °C. [lit.[73]: 95.0 – 95.5 °C]

1 H NMR (400 MHz, CDCl3) δ/ppm = 6.85 (d, J = 8.4 Hz, 1H, 5-H), 6.74 – 6.63 (m, 2H, 2-H,

6-H), 5.50 (s, 1H, OH), 4.64 (s, 1H, NH), 4.11 (q, J = 7.1 Hz, 2H, CH2CH3), 3.88 (s, 3H, OCH3),

3.40 (q, J = 7.1 Hz, 2H, CH2CH2NH), 2.74 (t, J = 7.1 Hz, 2H, CH2CH2NH), 1.23 (t, J = 7.1 Hz,

3H, CH2CH3).

13 C NMR (101 MHz, CDCl3) δ/ppm = 156.7 (CO), 146.7 (C-3), 144.4 (C-4), 130.8 (C-1), 121.5

(C-2 or 6), 114.6 (C-5), 111.4 (C-2 or 6), 60.9 (CH2CH3), 56.0 (OCH3), 42.4 (CH2CH2NH), 36.0

(CH2CH2NH), 14.8 (CH2CH3).

-1 IR (ATR) ṽmax/cm = 3276, 2979, 1688, 1516, 1237, 1033.

173 EXPERIMENTAL PART

- HRMS (ESI): calcd. for C12H16NO4 (M-H) 238.10848; found 238.10860.

Purity (HPLC): > 96% (λ = 210 nm).

Ethyl (3-methoxy-4-phenoxyphenethyl)carbamate (167)

Following general procedure I, carbamate 166 (2.00 g, 8.36 mmol, 1.0 eq.), phenylboronic acid

(168, 3.06 g, 25.1 mmol, 3.0 eq.), copper(II) acetate (1.67 g, 9.20 mmol, 1.1 eq.), NEt3

(2.90 mL, 20.9 mmol, 2.5 eq.) and pyridine (1.70 mL, 20.9 mmol, 2.5 eq.) in CH2Cl2 (40 mL) were used. The reaction was completed after 22 h and the product was purified by FCC (4:1 hexanes/acetone) to yield diaryl ether 167 (2.24 g, 8.36 mmol, 85%) as colorless solid.

Rf = 0.35 (4:1 hexanes/acetone). m.p.: 40 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.32 – 7.26 (m, 2H, 3’-H, 5’-H), 7.07 – 7.01 (m, 1H, 4’-H), 6.96 – 6.92 (m, 2H, 2’-H, 6’-H), 6.90 (d, J = 8.1 Hz, 1H, 5-H), 6.83 (d, J = 1.8 Hz, 1H, 2-H),

6.74 (dd, J = 8.1, 1.8 Hz, 1H, 6-H), 4.69 (s, 1H, NH), 4.12 (q, J = 7.2 Hz, 2H, CH2CH3), 3.83

(s, 3H, OCH3), 3.45 (q, J = 7.0 Hz, 2H, CH2CH2NH), 2.81 (t, J = 7.0 Hz, 2H, CH2CH2NH), 1.24

(t, J = 7.0 Hz, 3H, CH2CH3).

13 C NMR (101 MHz, CDCl3) δ/ppm = 158.1 (C-1’), 156.7 (CO), 151.6 (C-3), 143.8 (C-4), 135.7 (C-1), 129.6 (C-3’, C-5’), 122.6 (C-4’), 121.3 (C-5 or 6), 121.3 (C-5 or 6), 117.3 (C-2’, C-6’),

113.5 (C-2), 61.0 (CH2CH3), 56.2 (OCH3), 42.3 (CH2CH2NH), 36.2 (CH2CH2NH), 14.8

(CH2CH3).

-1 IR (ATR) ṽmax/cm = 3303, 2971, 1688, 1490, 1272, 1255, 1222, 1035, 754.

+ HRMS (ESI): calcd. for C18H22NO4 (M+H) 316.15433; found 316.15425.

Purity (HPLC): > 96% (λ = 210 nm).

174 EXPERIMENTAL PART

Ethyl (3,4-dimethoxyphenethyl)carbamate (169)

Following general procedure E, 3,4-dimethoxyphenylethylamine (161, 5.00 mL, 29.6 mmol, 1.0 eq.) in CH2Cl2 (30.0 mL), NEt3 (12.0 mL, 88.9 mmol, 3.0 eq.) and ethyl chloroformate (162, 5.70 mL, 59.3 mmol, 2.0 eq.) were used. The mixture was heated to reflux for 4 h and extraction yielded carbamate 169 (7.05 g, 27.8 mmol, 94%) as colorless solid. NMR data are in accordance with literature[160].

Rf = 0.25 (4:1 hexanes/acetone). m.p.: 63 °C.

1 H NMR (400 MHz, CDCl3) δ/ppm = 6.81 (d, J = 8.0 Hz, 1H, 5-H), 6.78 – 6.65 (m, 2H, 2-H, 6-

H), 4.65 (s, 1H, NH), 4.11 (q, J = 7.1 Hz, 2H, CH2CH3), 3.88 – 3.85 (m, 6H, 2x OCH3), 3.41 (q,

J = 6.8 Hz, 2H, CH2CH2NH), 2.75 (t, J = 6.8 Hz, 2H, CH2CH2NH), 1.23 (t, J = 7.1 Hz, 3H,

CH2CH3).

13 C NMR (101 MHz, CDCl3) δ/ppm = 156.7 (CO), 149.2 (C-3), 147.8 (C-4), 131.5 (C-1), 120.8

(C-2), 112.1 (C-6), 111.5 (C-5), 60.9 (CH2CH3), 56.1 (OCH3), 56.0 (OCH3), 42.3 (CH2CH2NH),

35.9 (CH2CH2NH), 14.8 (CH2CH3).

-1 IR (ATR) ṽmax/cm = 3372, 2935, 1697, 1514, 1258, 1234, 1139, 1027.

+ HRMS (ESI): calcd. for C13H20NO4 (M+H) 254.13868; found 354.13885.

Purity (HPLC): > 96% (λ = 210 nm).

175 EXPERIMENTAL PART

5.2.2.2 Synthesis of enol ethers

Ethyl (4-formylphenyl) carbonate (176)

Following general procedure E, 4-hydroxybenzaldehyde (175, 1.00 g, 8.19 mmol, 1.0 eq.) in

CH2Cl2 (10 mL), NEt3 (1.70 mL, 12.3 mmol, 1.5 eq.) and ethyl chloroformate (162, 860 µL, 9.01 mmol, 1.1 eq.) were used. The reaction was completed after 14 h and the product was purified by FCC (CH2Cl2) to yield aldehyde 176 (1.53 g, 7.90 mmol, 97%) as colorless solid.

Rf = 0.63 (CH2Cl2). m.p.: 121 °C.

1 H NMR (500 MHz, CDCl3) δ/ppm = 9.99 (s, 1H, CHO), 7.93 – 7.91 (m, 2H, 3-H, 5-H), 7.40 –

7.32 (m, 2H, 2-H, 6-H), 4.36 – 4.32 (m, 2H, CH2CH3), 1.41 – 1.38 (m, 3H, CH2CH3).

13 C NMR (126 MHz, CDCl3) δ/ppm = 190.9 (CHO), 155.7 (C-1), 152.9 (CO), 134.2 (C-4), 131.4

(C-3, C-5), 121.9 (C-2, C-6), 65.4 (CH2), 14.3 (CH3).

-1 IR (ATR) ṽmax/cm = 1757, 1668, 1602, 1254, 1210, 1159, 1055, 998, 840, 775.

•+ HRMS (EI): calcd. for C10H9O4 (M) 194.0579; found 194.0579.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

Ethyl (4-(2-methoxyvinyl)phenyl) carbonate (177)

Following general procedure F, (methoxymethyl)-triphenylphosphonium chloride (9.53 g, 27.8 mmol, 1.2 eq.) and LDA (20% in THF/ethylbenzene/heptane, 11.6 mL, 23.2 mmol, 1.0 eq.) in dry THF (50 mL) and aldehyde 176 (4.50 g, 23.2 mmol, 1.0 eq.) in dry THF (50 mL) were used. The mixture was stirred for 16 h and the product was purified by FCC (9:1

176 EXPERIMENTAL PART hexanes/EtOAc) to yield the desired cis/trans-enol ether 177 (3.30 g, 14.8 mmol, 64%) as colorless oil.

Rf = 0.28 (4:1 hexanes/EtOAc).

1 H NMR (cis/trans, 400 MHz, CDCl3) δ/ppm = 7.59 – 7.55 (m, 2H, cis 3-H, 5-H), 7.24 – 7.20 (m, 2H, trans 3-H, 5-H), 7.10 – 7.06 (m, 4H, cis/trans 2-H, 6-H), 7.00 (d, J = 13.0 Hz, 1H, trans CHCH-O), 6.13 (d, J = 7.0 Hz, 1H, cis CHCH-O), 5.79 (d, J = 13.0 Hz, 1H, trans CHCH-

O), 5.21 (d, J = 7.0 Hz, 1H, cis CHCH-O), 4.31 (qd, J = 7.1, 1.6 Hz, 4H, cis/trans CH2CH3),

3.78 (s, 3H, cis OCH3), 3.68 (s, 3H, trans OCH3), 1.38 (td, J = 7.1, 1.6 Hz, 6H, cis/trans CH2CH3).

13 C NMR (cis/trans, 126 MHz, CDCl3) δ/ppm = 153.9 (trans CO), 153.8 (cis CO), 149.2 (trans CHCH-O), 149.2 (trans C-1), 148.9 (cis C-1), 148.1 (cis CHCH-O), 134.5 (trans C-4), 134.0 (cis C-4), 129.3 (cis C-3, C-5), 126.1 (trans C-3, C-5), 121.3 (trans C-2, C-6), 120.8

(cis C-2, C-6), 104.9 (cis CHCH-O), 104.3 (trans CHCH-O), 64.9 (trans CH2CH3), 60.8

(cis CH2CH3), 56.7 (cis/trans CH2CH3), 14.4 (cis/trans CH2CH3).

Cis/trans-ratio according to 1H-NMR is 0.83.

•+ HRMS (EI): calcd. for C12H14O4 (M) 222.0887; found 222.0879.

1-(2-Methoxyvinyl)-4-phenoxybenzene (178)

Following general procedure F, (methoxymethyl)-triphenylphosphonium chloride (1.18 g, 3.43 mmol, 1.2 eq.) and LDA (20% in THF/ethylbenzene/heptane, 1.90 mL, 2.86 mmol, 1.0 eq.) in dry THF (10 mL) and 4-phenoxybenzaldehyde (500 µL, 2.86 mmol, 1.0 eq.) in dry THF (10 mL) were used. The mixture was stirred for 50 h and the product was purified by FCC (9:1 hexanes/EtOAc) to yield the desired cis/trans-enol ether 178 (384 mg, 1.70 mmol, 59%) as colorless oil.

Rf = 0.73 (4:1 hexanes/EtOAc).

1 H NMR (cis/trans, 400 MHz, CDCl3) δ/ppm = 7.60 – 7.53 (m, 2H, cis 3-H, 5-H), 7.36 – 7.28 (m, 4H, cis/trans Ph), 7.22 – 7.18 (m, 2H, trans 2-H, 6-H or 3-H, 5-H), 7.12 – 7.05 (m, 2H, cis/trans Ph), 7.02 – 6.91 (m, 9H, 4x cis/trans Ph, trans CHCH-O, cis/trans 2-H, 6-H or/and 3-

177 EXPERIMENTAL PART

H, 5-H), 6.11 (d, J = 7.0 Hz, 1H, cis CHCH-O), 5.81 (d, J = 13.0 Hz, 1H, trans CHCH-O), 5.22

(d, J = 7.0 Hz, 1H, cis CHCH-O), 3.78 (s, 3H, cis OCH3), 3.69 (s, 3H, trans OCH3).

13 C NMR (cis/trans, 101 MHz, CDCl3) δ/ppm = 157.8 (cis/trans qPh), 157.7 (cis/trans qPh), 155.2 (cis/trans C-1), 154.9 (cis/trans C-1), 148.5 (trans CHCH-O), 147.4 (cis CHCH-O), 131.8 (cis/trans C-4), 131.5 (cis/trans C-4), 129.9 (cis/trans Ph), 129.8 (cis/trans Ph), 129.7 (cis C-3, C-5), 126.5 (trans C-2, C-6 or C-3, C-5), 123.1 (cis/trans Ph), 123.0 (cis/trans Ph), 119.6 (cis/trans Ph or C-2, C-6 or C-3, C-5), 119.1 (cis/trans Ph or C-2, C-6 or C-3, C-5), 118.7 (cis/trans Ph or C-2, C-6 or C-3, C-5), 118.6 (cis/trans Ph or C-2, C-6 or C-3, C-5), 105.1

(cis CHCH-O), 104.5 (trans CHCH-O), 60.8 (cis OCH3), 56.7 (trans OCH3).

Cis/trans-ratio according to 1H-NMR is 0.75.

-1 IR (ATR) ṽmax/cm = 3036, 2935, 2831, 1488, 1234, 1150, 1092, 750, 691.

•+ HRMS (EI): calcd. for C15H14O2 (M) 226.0988; found 226.0987.

1-(Benzyloxy)-4-(2-methoxyvinyl)benzene (179)

Following general procedure F, (methoxymethyl)-triphenylphosphonium chloride (4.11 g, 12.0 mmol, 1.2 eq.) and LDA (20% in THF/ethylbenzene/heptane, 7.90 mL, 12.0 mmol, 1.2 eq.) in dry THF (50 mL) and 4-(benzyloxy)benzaldehyde (2.12 g, 9.99 mmol, 1.0 eq.) in dry THF (50 mL) were used. The mixture was stirred for 16 h and the product was purified by FCC (6:1 hexanes/EtOAc) to yield the desired cis/trans-enol ether 179 (2.04 g, 8.49 mmol, 85%) as yellow solid.

Rf = 0.68 (4:1 hexanes/EtOAc). m.p.: 62 °C.

1 H NMR (cis/trans, 400 MHz, CDCl3) δ/ppm = 7.53 – 7.49 (m, 2H, cis 3-H, 5-H), 7.45 – 7.29

(m, 10H, cis/trans PhCH2), 7.18 – 7.13 (m, 2H, trans 3-H, 5-H), 6.95 – 6.88 (m, 5H, cis/trans 2- H, 6-H, trans CHCH-O), 6.06 (d, J = 7.0 Hz, 1H, cis CHCH-O), 5.78 (d, J = 13.0 Hz, 1H, trans CHCH-O), 5.18 (d, J = 7.0 Hz, 1H, cis CHCH-O), 5.09 – 5.03 (m, 4H, cis/trans PhCH2),

3.76 (s, 3H, cis OCH3), 3.66 (s, 3H, trans OCH3).

13 C NMR (cis/trans, 101 MHz, CDCl3) δ/ppm = 157.2 (cis/trans C-1), 157.0 (cis/trans C-1), 147.8 (trans CHCH-O), 146.6 (cis CHCH-O), 137.3 (cis/trans qPh), 137.2 (cis/trans qPh),

178 EXPERIMENTAL PART

129.5 (cis C-3, C-5), 129.3 (cis/trans C-4), 129.2 (cis/trans C-4), 128.8 (cis/trans Ph), 128.7 (cis/trans Ph), 128.1 (cis/trans Ph), 128.0 (cis/trans Ph), 127.6 (cis/trans Ph), 126.3 (trans C- 3, C-5), 115.3 (trans C-2, C-6), 114.8 (cis C-2, C-6), 105.4 (cis CHCH-O), 104.7 (trans CHCH-

O), 70.2 (cis/trans PhCH2), 70.1 (cis/trans PhCH2), 60.6 (cis OCH3), 56.6 (trans OCH3).

Cis/trans-ratio according to 1H-NMR is 1.1.

•+ HRMS (EI): calcd. for C16H16O2 (M) 240.1145; found 240.1146.

5.2.2.3 N-Acyl Pictet-Spengler reaction

(±)-Ethyl 7-((ethoxycarbonyl)oxy)-1-(4-((ethoxycarbonyl)oxy)benzyl)-6-methoxy-3,4- dihy-droisoquinoline-2(1H)-carboxylate (180)

Following general procedure G, carbamate 165 (348 mg, 1.12 mmol, 1.0 eq.), enol ether 177

(248 mg, 1.12 mmol, 1.0 eq.) and TFA (840 µL, 11.2 mmol, 10 eq.) were dissolved in CH2Cl2 (15 mL). The reaction was completed after 18 h and the product was purified by FCC (5:1 hexanes/acetone) to yield compound 180 (452 mg, 0.901 mmol, 81%) as colorless oil.

Rf = 0.20 (4:1 hexanes/acetone).

1 H NMR (400 MHz, C2D2Cl4, 100 °C) δ/ppm = 7.03 (s, 4H, 2’-H, 3’-H, 5’-H, 6’-H), 6.69 – 6.63

(m, 2H, 5-H, 8-H), 5.19 (t, J = 6.6 Hz, 1H, 1-H), 4.28 – 4.22 (m, 4H, 2 OCOOCH2CH3), 4.05 –

3.92 (m, 3H, NCOOCH2CH3, 3-H), 3.79 (s, 3H, OCH3), 3.23 (ddd, J = 13.6, 9.7, 4.6 Hz, 1H, 3- H), 3.00 (h, J = 6.6 Hz, 2H, α-H), 2.81 (ddd, J = 15.8, 9.7, 5.8 Hz, 1H, 4-H), 2.61 – 2.54 (m,

1H, 4-H), 1.35 – 1.31 (m, 6H, 2 OCOOCH2CH3), 1.12 (t, J = 7.2 Hz, 3H, NCOOCH2CH3).

13 C NMR (101 MHz, C2D2Cl4, 100 °C) δ/ppm = 155.6 (N-CO), 153.6 (O-CO), 153.3 (O-CO), 150.4 (C-4’), 150.3 (C-6), 139.1 (C-7), 136.0 (C-1’), 133.4 (C-4a), 130.7 (C-2’, C-6’or C-3’, C- 5’), 129.3 (C-8a), 121.3 (C-5 or C-8), 120.9 (C-2’, C-6’ or C-3’, C-5’), 113.6 (C-5 or C-8), 65.1

179 EXPERIMENTAL PART

(OCOOCH2CH3), 64.9 (OCOOCH2CH3), 61.5 (NCOOCH2CH3), 56.6 (OCH3), 56.0 (C-1), 42.5

(C-α), 38.6 (C-3), 28.7 (C-4), 14.8 (NCOOCH2CH3), 14.4 (OCOOCH2CH3), 14.3

(OCOOCH2CH3).

-1 IR (ATR) ṽmax/cm = 2981, 2940, 1758, 1692, 1248, 1215, 1198.

+ HRMS (ESI): calcd. for C26H35N2O9 (M+NH4) 519.23371; found 519.23373.

Purity (HPLC): > 96% (λ = 210 nm).

(±)-Ethyl 1-(4-((ethoxycarbonyl)oxy)benzyl)-6,7-dimethoxy-3,4-dihydroisoquinoline- 2(1H)-carboxylate – SG-089 (181)

Following general procedure G, carbamate 169 (5.60 g, 22.1 mmol, 1.0 eq.), enol ether 177

(4.92 g, 22.1 mmol, 1.0 eq.) and TFA (18.0 mL, 243 mmol, 11 eq.) were dissolved in CH2Cl2 (50 mL). The reaction was completed after 20 h and the product was purified by FCC (5:1 hexanes/acetone) to give SG-089 181 (7.50 g, 16.9 mmol, 77%) as colorless solid.

Rf = 0.28 (4:1 hexanes/acetone). m.p.: 134 °C.

1 H NMR (400 MHz, C2D2Cl4, 100 °C) δ/ppm = 7.10 – 6.97 (m, 4H, 2’-H, 3’-H, 5’-H, 6’-H), 6.57 (s, 1H, 5-H), 6.23 (s, 1H, 8-H), 5.15 (t, J = 6.6 Hz, 1H, 1-H), 4.26 (q, J = 7.1 Hz, 2H,

OCOOCH2CH3), 4.13 – 3.98 (m, 2H, NCOOCH2CH3), 3.98 – 3.82 (m, 1H, 3-H), 3.78 (s, 3H, 6-

OCH3), 3.61 (s, 3H, 7-OCH3), 3.30 (ddd, J = 13.4, 9.4, 4.7 Hz, 1H, 3-H), 3.09 (dd, J = 13.4, 6.8 Hz, 1H, α-H), 2.94 (dd, J = 13.4, 6.8 Hz, 1H, α-H), 2.77 (ddd, J = 15.6, 9.4, 5.9 Hz, 1H, 4-H),

2.57 (dt, J = 15.6, 4.7 Hz, 1H, 4-H), 1.34 (t, J = 7.1 Hz, 3H, OCOOCH2CH3), 1.17 (t, J = 7.1

Hz, 3H, NCOOCH2CH3).

13 C NMR (101 MHz, C2D2Cl4, 100 °C) δ/ppm = 155.7 (N-CO), 153.6 (O-CO), 150.3 (C-4’), 148.8 (C-6), 148.1 (C-7), 136.4 (C-1’), 130.8 (C-2’, C-6’), 129.0 (C-8a), 127.0 (C-4a), 120.9 (C-

3’, C-5’), 113.3 (C-5), 112.3 (C-8), 64.9 (OCOOCH2CH3), 61.5 (NCOOCH2CH3), 56.7 (OCH3-

6), 56.6 (OCH3-7), 56.5 (C-1), 42.6 (C-α), 39.1 (C-3), 28.2 (C-4), 14.8 (OCOOCH2CH3), 14.4

(NCOOCH2CH3).

180 EXPERIMENTAL PART

-1 IR (ATR) ṽmax/cm = 3006, 2918, 2849, 1754, 1673, 1281, 1260, 1245, 1206, 1096, 859.

+ HRMS (ESI): calcd. for C24H30NO7 (M+H) 444.20168; found 444.20143.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

(±)-Ethyl 1-(4-((ethoxycarbonyl)oxy)benzyl)-6-methoxy-7-phenoxy-3,4-dihydroisoquino- line-2(1H)-carboxylate (182)

Following general procedure G, carbamate 167 (1.60 g, 5.06 mmol, 1.0 eq.), enol ether 177

(1.35 g, 6.07 mmol, 1.2 eq.) and TFA (3.80 mL, 50.6 mmol, 10 eq.) were dissolved in CH2Cl2 (50 mL). The reaction was completed after 90 h and the product was purified by FCC (5:1 hexanes/acetone) to yield compound 182 (2.30 g, 4.55 mmol, 90%) as yellow oil.

Rf = 0.31 (4:1 hexanes/acetone).

1 H NMR (400 MHz, C2D2Cl4, 100 °C) δ/ppm = 7.25 – 7.21 (m, 2H, Ar), 6.99 (s, 5H, Ph), 6.86 – 6.82 (m, 2H, Ar), 6.69 (s, 1H, 5-H), 6.54 (s, 1H, 8-H), 5.13 (t, J = 6.6 Hz, 1H, 1-H), 4.25

(q, J = 7.1 Hz, 2H, OCOOCH2CH3), 4.04 – 3.92 (m, 3H, NCOOCH2CH3, 3-H), 3.75 (s, 3H,

OCH3), 3.26 (ddd, J = 13.2, 9.6, 4.6 Hz, 1H, 3-H), 3.02 (dd, J = 13.7, 7.0 Hz, 1H, α-H), 2.94 (dd, J = 13.7, 7.0 Hz, 1H, α-H), 2.86 – 2.77 (m, 1H, 4-H), 2.60 (dt, J = 15.8, 4.6 Hz, 1H, 4-H),

1.33 (t, J = 7.0 Hz, 3H, OCOOCH2CH3), 1.12 (t, J = 7.2 Hz, 3H, NCOOCH2CH3).

13 C NMR (101 MHz, C2D2Cl4, 100 °C) δ/ppm = 158.5 (C-4’), 155.7 (N-CO), 153.5 (O-CO), 150.8 (C-6), 150.3 (C-7), 144.1 (C-8a), 136.0 (qPh), 131.2 (C-4a), 130.6 (Ph), 129.8 (C-1’), 129.6 (C-2’, C-6’ or C-3’, C-5’), 122.6 (Ph), 120.8 (Ph), 120.4 (C-8), 117.5 (C-2’, C-6’ or C-3’,

C-5’), 114.5 (C-6), 64.9 (OCOOCH2CH3), 61.5 (NCOOCH2CH3), 56.8 (OCH3), 56.0 (C-1), 42.5

(C-α), 38.9 (C-3), 28.5 (C-4), 14.8 (NCOOCH2CH3), 14.4 (OCOOCH2CH3).

-1 IR (ATR) ṽmax/cm = 2980, 1759, 1688, 1508, 1252, 1215, 1096, 747.

+ HRMS (ESI): calcd. for C29H32NO7 (M+H) 506.21733; found 506.21721.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

181 EXPERIMENTAL PART

(±)-Ethyl 7-(benzyloxy)-1-(4-(benzyloxy)benzyl)-6-methoxy-3,4-dihydroisoquinoline- 2(1H)-carboxylate - SG-145 (183)

Following general procedure G, carbamate 170 (861 mg, 2.61 mmol, 1.0 eq.), enol ether 179

(691 mg, 2.88 mmol, 1.1 eq.) and TFA (2.00 mL, 26.1 mmol, 10 eq.) were dissolved in CH2Cl2 (25 mL). The reaction was completed after 18 h and the product was purified by FCC (5:1 hexanes/acetone) to yield SG-145 (183, 444 mg, 0.825 mmol, 32%) as yellow solid.

Rf = 0.26 (4:1 hexanes/acetone). m.p.: 96 °C. [lit.[152]: 115-118 °C]

1 H NMR (400 MHz, C2D2Cl4, 100 °C) δ/ppm = 7.36 – 7.27 (m, 10H, Ph), 6.95 – 6.92 (m, 2H, 2’-H, 6’-H), 6.85 – 6.83 (m, 2H, 3’-H, 5’-H), 6.59 (s, 1H, 5-H), 6.37 (s, 1H, 8-H), 5.10 (t, J = 6.7

Hz, 1H, 1-H), 4.99 (s, 2H, PhCH2), 4.86 (s, 2H, PhCH2), 4.10 – 3.90 (m, 3H, CH2CH3, 3-H),

3.80 (s, 3H, OCH3), 3.28 – 3.20 (m, 1H, 3-H), 2.98 (dd, J = 13.6, 6.7 Hz, 1H, α-H), 2.86 (dd, J = 13.6, 6.7 Hz, 1H, α-H), 2.80 – 2.71 (m, 1H, 4-H), 2.56 – 2.50 (m, 1H, 4-H), 1.15 (t, J = 7.1

Hz, 3H, CH2CH3).

13 C NMR (101 MHz, C2D2Cl4, 100 °C) δ/ppm = 158.0 (C-4’), 155.7 (CO), 149.5 (C-6), 147.2 (C-7), 137.8 (qPh), 137.7 (qPh), 131.3 (C-1’), 131.0 (C-2’, C-6’), 129.5 (C-8a), 128.7 (Ph), 128.6 (Ph), 128.0 (Ph), 127.9 (C-4a), 127.7 (Ph), 127.6 (Ph), 115.4 (C-8 or C-3’, C-5’), 115.3

(C-8 or C-3’, C-5’), 113.9 (C-5), 72.2 (PhCH2), 70.7 (PhCH2), 61.4 (CH2CH3), 56.9 (OCH3),

56.4 (C-1), 42.3 (C-α), 38.9 (C-3), 28.3 (C-2), 14.8 (CH2CH3).

-1 IR (ATR) ṽmax/cm = 3007, 2940, 1673, 1510, 1254, 1236, 1224, 1200, 1093, 743, 697.

+ HRMS (ESI): calcd. for C34H36NO5 (M+H) 538.25880; found 538.25880.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

182 EXPERIMENTAL PART

(±)-Ethyl 6,7-dimethoxy-1-(4-phenoxybenzyl)-3,4-dihydroisoquinoline-2(1H)- carboxylate (184)

Following general procedure G, carbamate 169 (150 mg, 0.663 mmol, 1.0 eq.), enol ether 178

(168 mg, 0.663 mmol, 1.0 eq.) and TFA (500 µL, 6.63 mmol, 10 eq.) were dissolved in CH2Cl2 (5.0 mL). The reaction was completed after 6 h and the product was purified by FCC (4:1 hexanes/acetone) to yield compound 184 (181 mg, 0.405 mmol, 92%) as colorless oil that solidifies upon freezing.

Rf = 0.32 (4:1 hexanes/acetone). m.p.: 30 °C.

1 H NMR (400 MHz, C2D2Cl4, 100 °C) δ/ppm = 7.30 – 7.25 (m, 2H, Ar), 7.06 – 7.01 (m, 3H, Ph), 6.95 (dd, J = 8.7, 1.1 Hz, 2H, Ar), 6.90 – 6.87 (m, 2H, Ph), 6.57 (s, 1H, 5-H), 6.30 (s, 1H, 8-H),

5.17 (t, J = 6.9 Hz, 1H, 1-H), 4.05 (q, J = 7.1 Hz, 2H, CH2CH3), 4.00 – 3.89 (m, 1H, 3-H), 3.78

(s, 3H, 6-OCH3), 3.66 (s, 3H, 7-OCH3), 3.30 (ddd, J = 13.5, 9.6, 4.7 Hz, 1H, 3-H), 3.07 (dd, J = 13.5, 6.8 Hz, 1H, α-H), 2.94 (dd, J = 13.5, 6.8 Hz, 1H, α-H), 2.78 (ddd, J = 15.8, 8.9, 6.1 Hz,

1H, 4-H), 2.58 (dt, J = 15.0, 4.7 Hz, 1H, 4-H), 1.18 (t, J = 7.1 Hz, 3H, CH2CH3).

13 C NMR (101 MHz, C2D2Cl4, 100 °C) δ/ppm = 157.9 (C-4’), 156.2 (qPh), 155.7 (CO), 148.9 (C-6), 148.1 (C-7), 133.7 (C-1’), 131.2 (Ph), 129.9 (Ar), 129.3 (C-8a), 127.1 (C-4a), 123.3 (Ph),

119.1 (Ar or Ph), 119.0 (Ar or Ph), 113.3 (C-5), 112.5 (C-8), 61.4 (CH2CH3), 56.7 (6-OCH3 and

7-OCH3), 56.5 (C-1), 42.6 (C-α), 39.0 (C-3), 28.3 (C-4), 14.9 (CH2CH3).

-1 IR (ATR) ṽmax/cm = 2979, 1678, 1506, 1488, 1227, 1098, 694.

+ HRMS (ESI): calcd. for C27H30NO5 (M+H) 448.21185; found 448.21224.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

183 EXPERIMENTAL PART

5.2.2.4 Lithium alanate reductions of carbamates and deprotections of carbonates

(±)-1-(4-Hydroxybenzyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-7-ol ((±)-N-methylcoclaurine) – SG-132 (159)

Following general procedure H, carbamate 180 (2.58 g, 5.14 mmol, 1.0 eq.) in dry THF (40 mL) and LiAlH4 (1.35 g, 35.6 mmol, 7.0 eq.) in dry THF (40 mL) were used. The mixture was heated to reflux for 3 h to yield (±)-N-methylcoclaurine (159, 1.21 g, 4.04 mmol, 79%) as off-white solid.

Rf = 0.09 (9:1 CH2Cl2/MeOH). m.p.: 86 °C. [lit.[191]: 110-112 °C]

1 H NMR (500 MHz, CD3OD) δ/ppm = 6.92 – 6.87 (m, 2H, Ar), 6.70 – 6.65 (m, 2H, Ar), 6.63 (s,

1H, 5-H), 6.09 (s, 1H, 8-H), 3.80 (s, 3H, OCH3), 3.66 (dd, J = 7.5, 4.9 Hz, 1H, 1-H), 3.15 (ddd, J = 12.5, 9.4, 5.5 Hz, 1H, 3-H), 3.06 (dd, J = 13.8, 4.9 Hz, 1H, α-H), 2.85 (ddd, J = 15.8, 9.4, 6.2 Hz, 1H, 4-H), 2.75 – 2.69 (m, 2H, α-H and 3-H), 2.63 (dt, J = 16.3, 4.9 Hz, 1H, 4-H), 2.45

(s, 3H, NCH3).

13 C NMR (126 MHz, CD3OD) δ/ppm = 156.8 (C-4’), 147.8 (C-6), 145.0 (C-7), 131.7 (C-1’), 131.6 (Ar), 130.7 (C-8a), 125.5 (C-4a), 116.0 (Ar), 115.8 (C-8), 112.6 (C-5), 66.0 (C-1), 56.3

(OCH3), 47.5 (C-α), 42.6 (NCH3), 40.7 (C-3), 26.1 (C-4).

-1 IR (ATR) ṽmax/cm = 2929, 1609, 1513, 1445, 1251, 1112, 1022, 830.

+ HRMS (ESI): calcd. for C18H22NO3 (M+H) 300.15942; found 300.15932.

Purity (HPLC): > 96% (λ = 210 nm).

184 EXPERIMENTAL PART

(±)-4-((6,7-Dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)methyl)phenol ((±)-armepavine) – SG-121 (185) and (±)-4-(6,7-dimethoxy-2-methyl-1,2,3,4- tetrahydroisoquinolin-1-yl)phenol – SG-121-NP (189)

Following general procedure H, SG-089 (181, 7.50 g, 16.9 mmol, 1.0 eq.) in dry THF (90 mL) and LiAlH4 (7.06 g, 186 mmol, 11 eq.) in dry THF (40 mL) were used. The mixture was heated to reflux for 3 h and the product was purified by FCC (9:1 CH2Cl2/MeOH) to yield (±)-armepavine (185, 1.83 g, 5.86 mmol, 35%) as off-white solid. NMR data are in accordance with literature[192].

Rf = 0.34 (9:1 CH2Cl2/MeOH). m.p.: 76 °C.

1 H NMR (400 MHz, CD3OD) δ/ppm = 6.90 – 6.86 (m, 2H, 2’-H, 6’-H), 6.72 – 6.68 (m, 2H, 3’-H,

5’-H), 6.66 (s, 1H, 5-H or 8-H), 5.84 (s, 1H, 5-H or 8-H), 3.77 (s, 3H, OCH3), 3.72 (dd, J = 9.5,

4.3 Hz, 1H, 1-H), 3.42 (s, 3H, OCH3), 3.24 – 3.14 (m, 2H, α-H, 3-H), 2.95 – 2.87 (m, 1H, 4-H),

2.78 (ddd, J = 12.5, 6.3, 3.1 Hz, 1H, 3-H), 2.70 – 2.63 (m, 2H, α-H, 4-H), 2.52 (s, 3H, NCH3).

13 C NMR (101 MHz, CD3OD) δ/ppm = 157.0 (C-4’), 149.1 (C-6 or C-7), 147.5 (C-6 or C-7), 132.0 (C-2’, C-6’), 131.3 (C-1’), 129.8 (C-4a or C-8a), 126.4 (C-4a or C-8a), 116.1 (C-3’, C-5’),

113.0 (C-5 or C-8), 112.9 (C-5 or C-8), 66.0 (C-1), 56.3 (OCH3), 55.9 (OCH3), 47.0 (C-3), 42.4

(NCH3), 40.0 (C-α), 26.0 (C-4).

-1 IR (ATR) ṽmax/cm = 2979, 2901, 2834, 1509, 1251, 1226, 1100, 828.

+ HRMS (ESI): calcd. for C19H24NO3 (M+H) 314.17507; found 314.17502.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

Column chromatography furthermore yielded SG-121-NP (189, 1.03 g, 3.44 mmol, 20%) as light yellow solid. Due to the instability of the enol ether (177) and for the sake of time, purification was reduced to a minimum during up-scale reactions. This resulted in impurities in form of 4-hydroxybenzaldehyde (175) after the Wittig reaction. During the following N-acyl Pictet-Spengler reaction, a side product was formed that was carried over as impurity to the next step. Reduction with LiAlH4 then yielded SG-121-NP (189).

185 EXPERIMENTAL PART

Rf = 0.21 (9:1 CH2Cl2/MeOH). m.p.: 85 °C.

1 H NMR (400 MHz, CD3OD) δ/ppm = 7.23 – 7.13 (m, 2H, 2’,6’-H), 6.98 – 6.84 (m, 3H, 5,3’.5’-

H), 6.30 (s, 1H, 8-H), 5.49 (s, 1H, 1-H), 3.85 (s, 3H, OCH3), 3.74 – 3.62 (m, 1H, 3 or 4-H), 3.59

(s, 3H, OCH3), 3.50 – 3.43 (m, 1H, 3 or 4-H), 3.30 – 3.26 (m, 1H, 3 or 4-H), 3.23 – 3.13 (m,

1H, 3 or 4-H), 2.83 (s, 3H, NCH3).

13 C NMR (101 MHz, CD3OD) δ/ppm = 160.6 (C-4’), 150.9 (C-6 or C-7), 149.9 (C-6 or C-7), 133.3 (C-1’, C-2’ and/or C-6’), 133.2 (C-1’, C-2’ and/or C-6’), 133.0 (C-1’, C-2’ and/or C-6’), 124.8 (C-4a or C-8a), 124.7 (C-4a or C-8a), 117.0 (C-3’, C-5’), 112.2 (C-5 and C-8), 69.6 (C-

1), 56.4 (OCH3), 56.3 (OCH3), 41.2 (NCH3), 25.7 (C-3 or C-4), 25.6 (C-3 or C-4).

-1 IR (ATR) ṽmax/cm = 3132, 2980, 2725, 1615, 1516, 1270, 1229, 1105, 838.

+ HRMS (ESI): calcd. for C18H22NO3 (M+H) 300.15942; found 300.15964.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

(±)-4-((6-Methoxy-2-methyl-7-phenoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)methyl)phenol (186)

Following general procedure H, carbamate 182 (1.80 g, 3.56 mmol, 1.0 eq.) in dry THF (20 mL) and LiAlH4 (1.62 g, 42.7 mmol, 12 eq.) in dry THF (20 mL) were used. The mixture was heated to reflux for 5 h to yield racemic compound 186 (1.21 g, 4.04 mmol, 79%) as off-white solid.

Rf = 0.38 (9:1 CH2Cl2/MeOH). m.p.: 85 °C.

1 H NMR (500 MHz, CD3OD) δ/ppm = 7.26 – 7.20 (m, 2H, Ph), 6.99 – 6.96 (m, 1H, Ph), 6.84 – 6.80 (m, 3H, 5-H, 2’-H, 6’-H), 6.72 – 6.68 (m, 2H, Ph), 6.61 – 6.56 (m, 2H, 3’-H, 5’-H), 6.10

(s, 1H, 8-H), 3.73 (s, 3H, OCH3), 3.67 (dd, J = 8.6, 4.3 Hz, 1H, 1-H), 3.21 (ddd, J = 13.7, 9.3, 6.1 Hz, 1H, 3-H), 3.08 (dd, J = 13.5, 4.3 Hz, 1H, α-H), 2.98 – 2.91 (m, 1H, 4-H), 2.79 – 2.73

(m, 2H, 3-H and 4-H), 2.69 (dd, J = 13.5, 8.6 Hz, 1H, α-H), 2.50 (s, 3H, NCH3).

186 EXPERIMENTAL PART

13 C NMR (126 MHz, CD3OD) δ/ppm = 158.0 (qPh), 155.3 (C-4’), 149.9 (C-6), 142.0 (C-7), 130.3 (C-2’, C-6’), 129.9 (C-4a, C-8a or C-1’), 129.6 (C-4a, C-8a or C-1’), 129.4 (C-4a, C-8a or C-1’), 129.0 (Ph), 121.8 (Ph), 120.5 (C-8), 116.5 (Ph), 114.7 (C-3’, C-5’), 112.6 (C-5), 64.4

-1 (C-1), 54.9 (OCH3), 45.9 (C-3), 41.2 (NCH3), 38.6 (C-α), 25.1 (C-4). IR (ATR) ṽmax/cm = 2980, 2889, 1590, 1508, 1489, 1261, 1215, 749, 690.

-1 IR (ATR) ṽmax/cm = 2980, 2889, 1590, 1508, 1489, 1261, 1215, 749, 690.

+ HRMS (ESI): calcd. for C24H26NO3 (M+H) 376.19072; found 376.19069.

Purity (HPLC): > 96% (λ = 210 nm).

(±)-7-(Benzyloxy)-1-(4-(benzyloxy)benzyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydro- isoquinoline – SG-005 (187)

Following general procedure H, carbamate 183 (310 mg, 0.557 mmol, 1.0 eq.) in dry THF

(10 mL) and LiAlH4 (157 mg, 4.14 mmol, 7.2 eq.) in dry THF (20 mL) were used. The mixture was heated to reflux for 18 h and the product was purified by FCC (9:1 CH2Cl2/MeOH) to yield racemic amine SG-005 (187, 126 mg, 0.263 mmol, 46%) as colorless oil. The hydrochloride salt was formed according to general procedure J to yield a colorless solid. Analytical data are in accordance with literature[82].

Rf = 0.39 (9:1 CH2Cl2/MeOH). m.p.: 68 °C (HCl salt).

1 H NMR (500 MHz, CD3OD) δ/ppm = 7.37 – 7.22 (m, 10H, PhCH2), 6.96 – 6.93 (m, 2H, 2’-H, 6’-H), 6.93 – 6.90 (m, 2H, 3’-H, 5’-H), 6.68 (s, 1H, 5-H), 5.85 (s, 1H, 8-H), 4.99 (d, J = 2.3 Hz,

2H, 4’-OCH2), 4.60 – 4.54 (m, 2H, 7-OCH2), 3.78 (s, 3H, OCH3), 3.72 (dd, J = 9.4, 4.2 Hz, 1H, 1-H), 3.23 – 3.15 (m, 2H, 4-H, α-H), 2.91 (ddd, J = 16.3, 9.9, 6.3 Hz, 1H, 3-H), 2.78 (ddd, J =

12.5, 6.3, 3.2 Hz, 1H, 4-H), 2.71 – 2.65 (m, 2H, 3-H, α-H), 2.53 (s, 3H, NCH3).

13 C NMR (126 MHz, CD3OD) δ/ppm = 158.9 (C-4’), 149.7 (C-6), 146.7 (C-7), 138.7 (qPh), 138.6 (qPh), 132.9 (C-1’), 132.2 (C-2’, C-6’), 129.6 (C-8a), 129.5 (Ph), 129.4 (Ph), 128.9 (Ph), 128.8 (Ph), 128.6 (Ph), 127.1 (C-4a), 115.8 (C-3’, C-5’), 115.4 (C-8), 113.2 (C-5), 71.8

187 EXPERIMENTAL PART

(PhCH2), 71.0 (PhCH2), 65.9 (C-1), 56.4 (OCH3), 47.1 (C-4), 42.4 (NCH3), 39.7 (C-α), 26.2 (C- 3).

-1 IR (ATR) ṽmax/cm = 2932, 2850, 1608, 1509, 1454, 1222, 1099, 1013, 767, 696.

+ HRMS (ESI): calcd. for C32H34NO3 (M+H) 480.25332; found 480.25476.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

Separation of racemic SG-005 (187) via chiral HPLC (9:1 n-heptane/isopropanol + 0.45% diethylamine) yielded the two enantiomers (S)-SG-005 (209, colorless oil, retention time: ~ 10 min) and (R)-SG-005 (210, colorless oil, retention time: ~ 20 min).

(S)-SG-005 (209): [] = + 54.3 (c = 0.078, CHCl3).

[193] (R)-SG-005 (210): [] = - 65.4 (c = 0.052, CHCl3). (lit. : -37.6 (c = 0.9, CHCl3).

(±)-6,7-Dimethoxy-2-methyl-1-(4-phenoxybenzyl)-1,2,3,4-tetrahydroisoquinoline – SG-083 (188)

Following general procedure H, carbamate 184 (110 mg, 0.246 mmol, 1.0 eq.) in dry THF

(15 mL) and LiAlH4 (37.3 mg, 0.983 mmol, 4.0 eq.) in dry THF (10 mL) were used. The mixture was heated to reflux for 20 h and the product was purified by FCC (9.5:0.5 CH2Cl2/MeOH) to yield racemic amine SG-083 (188, 44.0 mg, 0.113 mmol, 46%) as light yellow oil.

Rf = 0.55 (9:1 CH2Cl2/MeOH).

1 H NMR (500 MHz, CD3OD) δ/ppm = 7.35 – 7.31 (m, 2H, Ph), 7.10 – 7.05 (m, 3H, Ph, 2’-H, 6’- H), 6.96 – 6.93 (m, 2H, Ph), 6.91 – 6.87 (m, 2H, 3’-H, 5’-H), 6.67 (s, 1H, 5-H), 5.97 (s, 1H, 8-

188 EXPERIMENTAL PART

H), 3.81 – 3.77 (m, 4H, 1-H, OCH3), 3.50 (s, 3H, OCH3), 3.25 – 3.18 (m, 2H, 3-H, α-H), 2.94 – 2.86 (m, 1H, 4-H), 2.81 – 2.75 (m, 2H, 3-H, α-H), 2.67 (ddd, J = 16.6, 5.4, 3.3 Hz, 1H, 4-H),

2.53 (s, 3H, NCH3).

13 C NMR (126 MHz, CD3OD) δ/ppm = 159.0 (qPh), 157.1 (C-4’), 149.2 (C-6), 147.7 (C-7), 135.9 (C-1’), 132.4 (C-2’, C-6’), 130.8 (Ph), 129.9 (C-8a), 126.9 (C-4a), 124.2 (Ph), 119.8 (C-

3’, C-5’), 119.6 (Ph), 113.1 (C-5 or C-8), 113.0 (C-5 or C-8), 65.9 (C-1), 56.4 (OCH3), 56.1

(OCH3), 47.1 (C-3), 42.5 (NCH3), 40.2 (C-α), 26.1 (C-4).

-1 IR (ATR) ṽmax/cm = 2938, 2833, 1589, 1505, 1488, 1228.

+ HRMS (ESI): calcd. for C25H28NO3 (M+H) 390.20637; found 390.20640.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

189 EXPERIMENTAL PART

5.2.2.5 Chan-Evans-Lam couplings

(±)-6-Methoxy-2-methyl-7-phenoxy-1-(4-phenoxybenzyl)-1,2,3,4-tetrahydroisoquinoline – SG-094 (192)

Following general procedure I, SG-132 (159, 150 mg, 0.500 mmol, 1.0 eq.), phenylboronic acid

(168, 366 mg, 3.00 mmol, 6.0 eq.), Cu(OAc)2 (200 mg, 1.10 mmol, 2.2 eq.), NEt3 (348 µL,

2.50 mmol, 5.0 eq.), pyridine (202 µL, 2.50 mmol, 5.0 eq.) and CH2Cl2 (50 mL) were used. The reaction was completed after 18 h and FCC (9.9:0.1 CH2Cl2/MeOH) yielded racemic diaryl ether SG-094 (192, 158 mg, 0.350 mmol, 70%) as colorless oil. The hydrochloride salt was formed according to general procedure J to yield a colorless solid.

Rf = 0.61 (9:1 CH2Cl2/MeOH). m.p.: 75 °C (HCl salt).

1 H NMR (400 MHz, CD3OD) δ/ppm = 7.29 – 7.18 (m, 4H, Ph), 7.07 – 7.00 (m, 3H, 2’-H, 6’-H, Ph), 6.96 – 6.92 (m, 1H, Ph), 6.86 – 6.82 (m, 3H, 5-H, Ph), 6.79 – 6.75 (m, 2H, 3’-H, 5’-H), 6.72 – 6.68 (m, 2H, Ph), 6.10 (s, 1H, 8-H), 3.75 (dd, J = 8.7, 4.4 Hz, 1H, 1-H), 3.72 (s, 3H,

OCH3), 3.28 – 3.14 (m, 2H, α-H, 3-H), 2.96 (ddd, J = 15.5, 9.1, 6.6 Hz, 1H, 4-H), 2.83 – 2.74

(m, 3H, α-H, 3-H, 4-H), 2.53 (s, 3H, NCH3).

13 C NMR (101 MHz, CD3OD) δ/ppm = 159.8 (qPh), 159.0 (qPh), 156.9 (C-4’), 151.7 (C-6), 143.0 (C-7), 135.5 (C-1’), 132.2 (C-2’, C-6’), 131.9 (C-4a or C-8a), 130.8 (Ph), 130.6 (C-4a or C-8a), 130.4 (Ph), 124.0 (Ph), 123.0 (Ph), 122.6 (C-8), 119.9 (C-3’, C-5’), 119.5 (Ph), 117.3

(Ph), 114.1 (C-5), 65.7 (C-1), 56.3 (OCH3), 47.3 (C-3), 42.6 (NCH3), 39.9 (C-α), 26.6 (C-4).

-1 IR (ATR) ṽmax/cm = 3038, 2937, 2838, 2793, 1588, 1505, 1487, 1218, 749, 690.

+ HRMS (ESI): calcd. for C30H30NO3 (M+H) 452.22202; found 452.22163.

190 EXPERIMENTAL PART

Purity (HPLC): > 96% (λ = 210 nm).

Separation of racemic SG-094 (192) via chiral HPLC (9.5:0.5 n-heptane/isopropanol + 0.45% diethylamine) yielded the two enantiomers (S)-SG-094 (211, colorless oil, retention time: ~ 8 min) and (R)-SG-094 (212, colorless oil, retention time: ~ 11 min).

(S)-SG-094 (211): [] = + 74.5 (c = 0.043, CHCl3).

(R)-SG-094 (212): [] = - 52.5 (c = 0.057, CHCl3).

(±)-6,7-Dimethoxy-1-(4-(4-methoxyphenoxy)benzyl)-2-methyl-1,2,3,4-tetrahydro- isoquinoline – SG-122 (193)

Following general procedure I, SG-121 (185, 157 mg, 0.500 mmol, 1.0 eq.), 4-methoxyphenyl- boronic acid (228 mg, 1.50 mmol, 3.0 eq.), Cu(OAc)2 (100 mg, 0.550 mmol, 1.1 eq.), NEt3

(174 µL, 1.25 mmol, 2.5 eq.), pyridine (101 µL, 1.25 mmol, 2.5 eq.) and CH2Cl2 (50 mL) were used. After 18 h the reaction was completed and the product was purified by FCC (9.5:0.5

CH2Cl2/MeOH) to yield racemic diaryl ether SG-122 (193, 185 mg, 0.441 mmol, 88%) as colorless oil. The hydrochloride salt was formed according to general procedure J to yield a colorless solid.

Rf = 0.45 (9:1 CH2Cl2/MeOH). m.p.: 204 °C (HCl salt).

1 H NMR (500 MHz, CDCl3) δ/ppm = 7.04 – 7.01 (m, 2H, 2’-H, 6’-H), 6.96 – 6.92 (m, 2H, Ar),

6.88 – 6.83 (m, 4H, 2’-H, 6’-H, Ar), 6.56 (s, 1H, 5-H), 6.05 (s, 1H, 8-H), 3.84 (s, 3H, OCH3),

191 EXPERIMENTAL PART

3.80 (s, 3H, OCH3), 3.69 (dd, J = 7.5, 5.2 Hz, 1H, 1-H), 3.60 (s, 3H, OCH3), 3.23 – 3.12 (m, 2H, α-H, 3-H), 2.86 – 2.74 (m, 3H, α-H, 3-H, 4-H), 2.58 (dt, J = 15.6, 4.5 Hz, 1H, 4-H), 2.53 (s,

3H, NCH3).

13 C NMR (126 MHz, CDCl3) δ/ppm = 156.9 (C-4’), 155.9 (qAr), 150.6 (qAr), 147.4 (C-6), 146.4 (C-7), 134.3 (C-1’), 131.0 (C-2’, C-6’), 129.4 (C-8a), 126.1 (C-4a), 120.7 (Ar), 117.6 (C-3’, C-

5’ or Ar), 115.0 (C-3’, C-5’ or Ar), 111.3 (C-5), 111.2 (C-8), 65.0 (C-1), 55.9 (OCH3), 55.8

(OCH3), 55.7 (OCH3), 47.0 (C-3), 42.8 (NCH3), 40.6 (C-α), 25.6 (C-4).

-1 IR (ATR) ṽmax/cm = 2971, 2629, 1504, 1265, 1227, 1111, 1006, 845.

+ HRMS (ESI): calcd. for C26H30NO4 (M+H) 420.21693; found 420.21657.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

(±)-6,7-Dimethoxy-2-methyl-1-(4-(3,4,5-trimethoxyphenoxy)benzyl)-1,2,3,4-tetrahydro- isoquinoline hydrochloride – SG-127 (194)

Following general procedure I, SG-121 (185, 157 mg, 0.500 mmol, 1.0 eq.), 3,4,5-trimethoxy- phenylboronic acid (318 mg, 1.50 mmol, 3.0 eq.), Cu(OAc)2 (100 mg, 0.550 mmol, 1.1 eq.),

NEt3 (174 µL, 1.25 mmol, 2.5 eq.), pyridine (101 µL, 1.25 mmol, 2.5 eq.) and CH2Cl2 (50 mL) were used. After 18 h the reaction was completed and the product was purified by FCC (CH2Cl2

+ 1% NEt3), followed by recrystallization in EtOH as hydrochloride salt to yield diaryl ether SG- 127 (194, 216 mg, 0.418 mmol, 84%) as colorless solid. Analytical data are related to the hydrochloride salt.

Rf = 0.47 (9:1 CH2Cl2/MeOH). m.p.: 183 °C (HCl salt).

1 H NMR (500 MHz, CDCl3) δ/ppm = 13.13 (s, 1H, NH), 7.11 – 7.05 (m, 2H, 2’-H, 6’-H), 6.94 – 6.90 (m, 2H, 3’-H, 5’-H), 6.64 (s, 1H, 5-H), 6.22 (s, 2H, 2’’-H, 6’’-H), 5.71 (s, 1H, 8-H), 4.21 –

4.11 (m, 2H, 1-H, α-H), 3.86 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.78 (s, 6H, 2 OCH3), 3.76 –

3.70 (m, 1H, 3-H), 3.50 (s, 3H, OCH3), 3.40 – 3.33 (m, 1H, 3-H), 3.12 – 3.04 (m, 2H, 4-H), 2.94

– 2.91 (m, 1H, α-H), 2.89 (s, 3H, NCH3).

192 EXPERIMENTAL PART

13 C NMR (126 MHz, CDCl3) δ/ppm = 157.2 (C-4’), 154.1 (C-3’’, C-5’’), 152.9 (C-1’’), 149.5 (C-6), 147.5 (C-7), 134.6 (C-4’’), 131.8 (C-2’, C-6’), 130.2 (C-1’), 120.9 (C-4a or C-8a), 120.6 (C-4a or C-8a), 118.5 (C-3’, C-5’), 111.3 (C-5 or C-8), 111.2 (C-5 or C-8), 97.1 (C-2’’, C-6’’),

65.8 (C-1), 61.2 (OCH3), 56.3 (2 OCH3), 56.1 (OCH3), 55.7 (OCH3), 44.5 (C-3), 41.0 (C-α),

40.1 (NCH3), 21.5 (C-4).

-1 IR (ATR) ṽmax/cm = 2970, 2834, 2441, 1496, 1221, 1126, 1110, 1009, 989, 857.

+ HRMS (ESI): calcd. for C28H34NO6 (M+H) 480.23806; found 480.23789.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

(±)-6,7-Dimethoxy-1-(4-(4-methoxyphenoxy)phenyl)-2-methyl-1,2,3,4-tetrahydro- isoquinoline – SG-157 (195)

Following general procedure I, SG-121-NP (189, 150 mg, 0.500 mmol, 1.0 eq.), 4-methoxy- phenylboronic acid (228 mg, 1.50 mmol, 3.0 eq.), Cu(OAc)2 (100 mg, 0.550 mmol, 1.1 eq.),

NEt3 (174 µL, 1.25 mmol, 2.5 eq.), pyridine (101 µL, 1.25 mmol, 2.5 eq.) and CH2Cl2 (50 mL) were used. After 18 h the reaction was completed and the product was purified by FCC (CH2Cl2

→ 9.8:0.2 CH2Cl2/MeOH) to yield diaryl ether SG-157 (195, 192 mg, 0.473 mmol, 95%) as colorless oil. The hydrochloride salt was formed according to general procedure J to yield a beige solid.

Rf = 0.49 (9:1 CH2Cl2/MeOH). m.p.: 97 °C (HCl salt).

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 7.22 – 7.16 (m, 2H, 2’-H, 6’-H), 7.03 – 6.92 (m, 4H, 2’’-H, 3’’-H, 5’’-H, 6’’-H), 6.90 – 6.84 (m, 2H, 3’-H, 5’-H), 6.69 (s, 1H, 5-H), 6.08 (s, 1H, 8-H),

4.15 (s, 1H, 1-H), 3.74 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.46 (s, 3H, OCH3), 3.02 – 2.95 (m,

2H, 3-H, 4-H), 2.71 – 2.63 (m, 1H, 4-H), 2.49 – 2.44 (m, 1H, 3-H), 2.12 (s, 3H, NCH3).

193 EXPERIMENTAL PART

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 156.9 (C-4’), 155.5 (C-4’’), 149.5 (C-1’’), 147.2 (C-6), 146.7 (C-7), 138.2 (C-1’), 130.4 (C-2’, C-6’), 130.1 (C-8a), 126.4 (C-4a), 120.6 (C-2’’, C-6’’ or C-3’’, C-5’’), 116.9 (C-3’, C-5’), 115.0 (C-2’’, C-6’’ or C-3’’, C-5’’), 111.8 (C-8), 111.4 (C-5), 69.0

(C-1), 55.5 (OCH3), 55.4 (2 OCH3), 51.2 (C-3), 43.8 (NCH3), 28.4 (C-4).

-1 IR (ATR) ṽmax/cm = 2970, 2903, 2837, 1514, 1263, 1225, 1025, 820, 744.

+ HRMS (ESI): calcd. for C25H28NO4 (M+H) 406.20128; found 406.20099.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

(±)-6-Methoxy-2-methyl-7-phenoxy-1-(4-(4-(trifluoromethyl)phenoxy)benzyl)-1,2,3,4- tetrahydroisoquinoline – SG-153 (199)

Following general procedure I, phenol 186 (150 mg, 0.400 mmol, 1.0 eq.), 4-(trifluoromethyl)- phenylboronic acid (228 mg, 1.20 mmol, 3.0 eq.), Cu(OAc)2 (79.8 mg, 0.440 mmol, 1.1 eq.),

NEt3 (139 µL, 1.00 mmol, 2.5 eq.), pyridine (080.8 µL, 1.00 mmol, 2.5 eq.) and CH2Cl2 (50 mL) were used. After 18 h the reaction was completed and the product was purified by FCC (CH2Cl2

→ 9.5:0.5 CH2Cl2/MeOH) to yield diaryl ether SG-153 (199, 50.7 mg, 0.0976 mmol, 24%) as brown oil. The hydrochloride salt was formed according to general procedure J to yield a yellow solid.

Rf = 0.50 (9:1 CH2Cl2/MeOH). m.p.: 92 °C (HCl salt).

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.54 – 7.48 (m, 2H, 3’’-H, 5’’-H), 7.26 – 7.22 (m, 2H, Ph), 7.08 – 7.04 (m, 2H, 2’-H, 6’-H), 7.01 – 6.92 (m, 3H, 2’’-H, 6’’-H, Ph), 6.87 – 6.79 (m, 4H, 3’-H,

5’-H, Ph), 6.68 (s, 1H, 5-H), 6.36 (s, 1H, 8-H), 3.77 (s, 3H, OCH3), 3.69 (t, J = 6.0 Hz, 1H, 1- H), 3.18 (ddd, J = 13.0, 7.8, 5.2 Hz, 1H, 3-H), 3.09 (dd, J = 13.3, 5.2 Hz, 1H, α-H), 2.90 – 2.82 (m, 2H, α-H, 4-H), 2.80 – 2.74 (m, 1H, 3-H), 2.66 (dt, J = 15.7, 5.2 Hz, 1H, 4-H), 2.53 (s, 3H,

NCH3).

13 C NMR (101 MHz, CDCl3) δ/ppm = 160.9 (C-1’’), 158.4 (qPh), 153.8 (C-4’), 150.0 (C-6),

142.2 (C-7), 136.0 (C-1’), 131.4 (C-2’, C-6’), 130.1 (C-8a), 129.6 (Ph), 128.7 (q, JCF = 277.9

194 EXPERIMENTAL PART

Hz, CF3), 127.2 (q, JCF = 3.6 Hz, C-4’’), 124.6 (q, JCF = 32.9 Hz, C-3’’, C-5’’), 124.4 (C-4a), 122.3 (Ph), 121.0 (C-8), 119.8 (C-3’, C-5’), 117.6 (C-2’’, C-6’’), 116.7 (Ph), 112.8 (C-5), 64.6

(C-1), 56.1 (OCH3), 47.4 (C-3), 43.0 (NCH3), 40.0 (C-α), 26.4 (C-4).

-1 IR (ATR) ṽmax/cm = 2980, 2907, 1602, 1506, 1325, 1243, 1221, 1103, 1064, 749.

+ HRMS (ESI): calcd. for C31H29F3NO3 (M+H) 520.20940; found 520.20907.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

(±)-6-Methoxy-2-methyl-7-phenoxy-1-(4-(4-(trifluoromethoxy)phenoxy)benzyl)-1,2,3,4- tetrahydroisoquinoline – SG-154 (198)

Following general procedure I, phenol 186 (150 mg, 0.400 mmol, 1.0 eq.), 4-

(trifluoromethoxy)-phenylboronic acid (247 mg, 1.20 mmol, 3.0 eq.), Cu(OAc)2 (79.8 mg,

0.440 mmol, 1.1 eq.), NEt3 (139 µL, 1.00 mmol, 2.5 eq.), pyridine (80.8 µL, 1.00 mmol, 2.5 eq.) and CH2Cl2 (20 mL) were used. After 13 h the reaction was completed and purification of the product via preparative HPLC (7:3 n-heptane/isopropanol + 0.45% diethylamine, 15 mL flow) yielded diaryl ether SG-154 (198, 99.6 mg, 0.186 mmol, 47%) as light yellow oil. The hydrochloride salt was formed according to general procedure J to yield a light yellow solid.

Rf = 0.51 (9:1 CH2Cl2/MeOH). m.p.: 90 °C (HCl salt).

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.28 – 7.21 (m, 2H, Ph, collapses with chloroform), 7.15 – 7.10 (m, 2H, 3’’-H, 5’’-H), 7.05 – 6.97 (m, 3H, 2’-H, 6’-H, Ph), 6.93 – 6.87 (m, 2H, 2’’-H, 6’’-H), 6.85 – 6.79 (m, 4H, 3’-H, 5’-H, Ph), 6.67 (s, 1H, 5-H), 6.36 (s, 1H, 8-H), 3.77 (s, 3H,

OCH3), 3.67 (t, J = 5.8 Hz, 1H, 1-H), 3.18 (ddd, J = 12.4, 8.0, 5.0 Hz, 1H, 3-H), 3.07 (dd, J = 13.8, 5.8 Hz, 1H, α-H), 2.90 – 2.81 (m, 2H, 4-H, α-H), 2.76 (dt, J = 12.4, 5.2 Hz, 1H, 3-H), 2.65

(dt, J = 15.9, 4.9 Hz, 1H, 4-H), 2.53 (s, 3H, NCH3).

13 C NMR (101 MHz, CDCl3) δ/ppm = 158.4 (qPh), 156.4 (C-1’’), 154.8 (C-4’), 150.0 (C-6),

144.3 (q, JCF = 4.1 Hz, C-4’’), 142.2 (C-7), 135.3 (C-1’), 131.3 (C-8a), 131.2 (C-2’, C-6’), 130.2

(C-4a), 129.5 (Ph), 122.6 (C-3’’, C-5’’), 122.2 (Ph), 121.0 (C-8), 120.7 (q, JCF = 256.5 Hz, CF3),

195 EXPERIMENTAL PART

119.2 (C-3’, C-5’ or C-2’’, C-6’’), 119.1 (C-3’, C-5’ or C-2’’, C-6’’), 116.7 (Ph), 112.8 (C-5), 64.6

(C-1), 56.1 (OCH3), 47.4 (C-3), 43.0 (NCH3), 40.0 (C-α), 26.4 (C-4).

-1 IR (ATR) ṽmax/cm = 2980, 2889, 2440, 1510, 1497, 1239, 1218, 1187, 1163, 1106, 853, 751.

+ HRMS (ESI): calcd. for C31H29F3NO4 (M+H) 536.20432; found 536.20393.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

(±)-6-Methoxy-1-(4-(4-methoxyphenoxy)benzyl)-2-methyl-7-phenoxy-1,2,3,4-tetrahydro- isoquinoline – SG-155 (196)

Following general procedure I, phenol 186 (188 mg, 0.500 mmol, 1.0 eq.), 4-methoxy- phenylboronic acid (228 mg, 1.50 mmol, 3.0 eq.), Cu(OAc)2 (100 mg, 0.550 mmol, 1.1 eq.),

NEt3 (174 µL, 1.25 mmol, 2.5 eq.), pyridine (101 µL, 1.25 mmol, 2.5 eq.) and CH2Cl2 (20 mL) were used. After 20 h the reaction was completed and the product was purified by FCC (CH2Cl2

→ 9.9:0.1 CH2Cl2/MeOH) to give diaryl ether SG-155 (196, 162 mg, 0.336 mmol, 67%) as colorless oil. The hydrochloride salt was formed according to general procedure J to yield a light yellow solid.

Rf = 0.49 (9:1 CH2Cl2/MeOH). m.p.: 81 °C (HCl salt).

1 H NMR (500 MHz, CD3OD) δ/ppm = 7.23 – 7.20 (m, 2H, Ph), 6.98 – 6.94 (m, 3H, 3’-H, 5’-H, Ph), 6.85 – 6.79 (m, 5H, 5-H, 2’’-H, 6’’-H, 3’’-H, 5’’-H), 6.72 – 6.68 (m, 4H, 2’-H, 6’-H, 3’-H, 5’-

H), 6.07 (s, 1H, 8-H), 3.77 (s, 3H, OCH3), 3.71 (s, 4H, 1-H, OCH3), 3.23 (ddd, J = 13.7, 9.4, 6.1 Hz, 1H, 3-H), 3.15 (dd, J = 13.3, 4.4 Hz, 1H, α-H), 2.97 (ddd, J = 15.9, 9.4, 6.8 Hz, 1H, 4-H),

2.82 – 2.73 (m, 3H, 3-H, 4-H, α-H), 2.53 (s, 3H, NCH3).

13 C NMR (126 MHz, CD3OD) δ/ppm = 159.8 (qPh), 158.2 (C-4’), 157.2 (C-4’’), 151.9 (C-6 or C-1’’), 151.7 (C-6 or C-1’’), 142.9 (C-7), 134.6 (C-1’), 132.0 (C-3’, C-5’), 131.8 (C-8a), 130.6 (C-4a), 130.4 (Ph), 123.0 (Ph), 122.6 (C-8), 121.4 (C-2’’, C-6’’ or C-3’’, C-5’’), 118.7 (C-2’, C-

6’), 117.3 (Ph), 115.9 (C-2’’, C-6’’ or C-3’’, C-5’’), 114.1 (C-5), 65.7 (C-1), 56.3 (OCH3), 56.1

(OCH3), 47.3 (C-3), 42.6 (NCH3), 39.8 (C-α), 26.6 (C-4).

196 EXPERIMENTAL PART

-1 IR (ATR) ṽmax/cm = 2940, 2907, 2523, 2360, 1496, 1264, 1218, 750, 690.

+ HRMS (ESI): calcd. for C31H32NO4 (M+H) 482.23258; found 482.23255.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

(±)-6-Methoxy-2-methyl-7-phenoxy-1-(4-(3,4,5-trimethoxyphenoxy)benzyl)-1,2,3,4-tetra- hydroisoquinoline – SG-161 (197)

Following general procedure I, phenol 186 (100 mg, 0.266 mmol, 1.0 eq.), 3,4,5-trimethoxy- phenylboronic acid (169 mg, 0.799 mmol, 3.0 eq.), Cu(OAc)2 (53.2 mg, 0.293 mmol, 1.1 eq.),

NEt3 (92.8 µL, 0.666 mmol, 2.5 eq.), pyridine (53.9 µL, 0.666 mmol, 2.5 eq.) and CH2Cl2 (50 mL) were used. After 20 h the reaction was completed and the product was purified by

FCC (CH2Cl2 → 9.7:0.3 CH2Cl2/MeOH) to yield diaryl ether SG-161 (197, 100 mg, 0.185 mmol, 69%) as colorless oil. The hydrochloride salt was formed according to general procedure J to yield a colorless solid.

Rf = 0.65 (9:1 CH2Cl2/MeOH). m.p.: 82 °C (HCl salt).

1 H NMR (500 MHz, CD3OD) δ/ppm = 7.23 – 7.18 (m, 2H, Ph), 7.04 – 7.00 (m, 2H, 2’-H, 6’-H), 6.96 – 6.92 (m, 1H, Ph), 6.83 (s, 1H, 5-H), 6.80 – 6.76 (m, 2H, 3’-H, 5’-H), 6.71 – 6.66 (m, 2H,

Ph), 6.20 (s, 2H, 2’’-H, 6’’-H), 6.14 (s, 1H, 8-H), 3.76 – 3.74 (m, 1H, 1-H), 3.73 (s, 3H, OCH3),

3.71 (s, 3H, OCH3), 3.70 (s, 6H, 2 OCH3), 3.24 (ddd, J = 13.7, 9.1, 6.1 Hz, 1H, 3-H), 3.16 (dd, J = 13.4, 4.4 Hz, 1H, α-H), 2.99 – 2.92 (m, 1H, 4-H), 2.83 – 2.75 (m, 3H, 3-H, 4-H, α-H), 2.54

(s, 3H, NCH3).

13 C NMR (126 MHz, CD3OD) δ/ppm = 159.8 (qPh), 157.1 (C-4’), 155.4 (C-1’’), 155.2 (C-3’’, C- 5’’), 151.7 (C-6), 143.0 (C-7), 135.4 (C-1’), 135.0 (C-4’’), 132.2 (C-2’, C-6’), 132.0 (C-8a), 130.6 (C-4a), 130.4 (Ph), 123.0 (Ph), 122.6 (C-8), 119.6 (C-3’, C-5’), 117.3 (Ph), 114.1 (C-5), 97.6

(C-2’’, C-6’’), 65.7 (C-1), 61.2 (OCH3), 56.5 (2 OCH3), 56.3 (OCH3), 47.5 (C-3), 42.7 (NCH3), 39.9 (C-α), 26.7 (C-4).

-1 IR (ATR) ṽmax/cm = 3725, 2940, 2360, 2341, 1496, 1216, 1124, 993, 690.

197 EXPERIMENTAL PART

+ HRMS (ESI): calcd. for C33H36NO6 (M+H) 542.25371; found 542.25327.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

(±)-6-Methoxy-2-methyl-7-phenoxy-1-(4-(p-tolyloxy)benzyl)-1,2,3,4- tetrahydroisoquinoline hydrochloride – SG-162 (200)

45 O 6 4a 3 HCl 1 N O 7 8a 2 8 5'' 5' 4'' 6'' 6' 1' 1'' 4' 3'' 2' 2'' O 3' 200 SG-162 ( )

Following general procedure I, phenol 186 (100 mg, 0.266 mmol, 1.0 eq.), 4-methylbenzene- boronic acid (109 mg, 0.799 mmol, 3.0 eq.), Cu(OAc)2 (53.2 mg, 0.293 mmol, 1.1 eq.), NEt3

(92.8 µL, 0.666 mmol, 2.5 eq.), pyridine (53.9 µL, 0.666 mmol, 2.5 eq.) and CH2Cl2 (20 mL) were used. After 20 h the reaction was completed and the product was purified by FCC (CH2Cl2

→ 9.7:0.3 CH2Cl2/MeOH) to yield diaryl ether SG-162 (200, 59.0 mg, 0.127 mmol, 48%) as light brown oil, which was, according to general procedure J, directly transferred into its hydrochloride salt to yield a beige solid. Analytical data are related to the hydrochloride salt.

Rf = 0.61 (9:1 CH2Cl2/MeOH). m.p.: 79 °C (HCl salt).

1 H NMR (500 MHz, CD3OD) δ/ppm = 7.27 – 7.23 (m, 2H, Ph), 7.12 – 7.06 (m, 4H, 2’-H, 6’-H, 2’’-H, 6’’-H), 7.03 – 6.98 (m, 2H, 5-H, Ph), 6.84 – 6.81 (m, 2H, 3’-H, 5’-H), 6.79 – 6.74 (m, 2H, 3’’-H, 5’’-H), 6.72 – 6.68 (m, 2H, Ph), 6.07 (s, 1H, 8-H), 4.56 (dd, J = 10.0, 4.7 Hz, 1H, 1-H),

3.90 – 3.83 (m, 1H, 3-H), 3.78 (s, 3H, OCH3), 3.54 – 3.48 (m, 1H, 3-H), 3.39 (dd, J = 13.2, 4.7

Hz, 1H, α-H), 3.28 – 3.20 (m, 2H, 4-H), 3.11 – 2.99 (m, 4H, α-H, NCH3), 2.32 (s, 3H, CH3).

13 C NMR (126 MHz, CD3OD) δ/ppm = 159.0 (qPh), 158.8 (C-4’), 155.8 (C-4’’), 153.3 (C-6), 144.4 (C-7), 134.4 (C-1’’), 132.3 (C-2’’, C-6’’), 131.3 (C-2’, C-6’), 130.6 (Ph), 130.4 (C-1’), 127.7 (C-4a), 123.6 (C-8), 122.3 (C-8a), 120.2 (C-3’’, C-5’’), 119.6 (C-3’, C-5’), 117.8 (Ph), 114.2 (C-

5), 65.7 (C-1), 57.5 (C-α), 56.4 (OCH3), 46.5 (C-3), 40.7 (NCH3), 24.7 (C-4), 20.7 (CH3).

-1 IR (ATR) ṽmax/cm = 3406, 2971, 2443, 1499, 1230, 1107, 749.

+ HRMS (ESI): calcd. for C31H32NO3 (M+H) 466.23767; found 466.23751.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

198 EXPERIMENTAL PART

(±)-6-Methoxy-2-methyl-7-phenoxy-1-(4-(pyridin-4-yloxy)benzyl)-1,2,3,4-tetrahydro- isoquinoline – SG-163 (203)

Following general procedure I, phenol 186 (100 mg, 0.266 mmol, 1.0 eq.), pyridine-4-boronic acid (98.2 mg, 0.799 mmol, 3.0 eq.), Cu(OAc)2 (53.2 mg, 0.293 mmol, 1.1 eq.), NEt3 (92.8 µL,

0.666 mmol, 2.5 eq.), pyridine (53.9 µL, 0.666 mmol, 2.5 eq.) and CH2Cl2 (20 mL) were used. After 20 h the reaction was completed and the product was purified via preparative HPLC (6:4 n-heptane/isopropanol + 0.45% diethylamine, 15 mL flow) to yield diaryl ether SG-163 (203, 30.6 mg, 0.0676 mmol, 25%) as colorless oil. The hydrochloride salt was formed according to general procedure J to yield a colorless solid.

Rf = 0.54 (9:1 CH2Cl2/MeOH). m.p.: 233 °C (HCl salt).

1 H NMR (400 MHz, CDCl3) δ/ppm = 8.41 (dd, J = 4.8, 1.5 Hz, 2H, 2’’-H, 6’’-H), 7.27 – 7.22 (m, 2H, Ph, collapses with chloroform), 7.11 – 7.07 (m, 2H, 2’-H, 6’-H), 7.01 – 6.96 (m, 1H, Ph), 6.90 – 6.86 (m, 2H, 3’-H, 5’-H), 6.85 – 6.82 (m, 2H, Ph), 6.76 – 6.72 (m, 2H, 3’’-H, 5’’-H), 6.68

(s, 1H, 5-H), 6.37 (s, 1H, 8-H), 3.77 (s, 3H, OCH3), 3.70 (t, J = 6.2 Hz, 1H, 1-H), 3.22 – 3.15 (m, 1H, 3-H), 3.10 (dd, J = 14.0, 5.4 Hz, 1H, α-H), 2.90 – 2.75 (m, 3H, 3-H, 4-H, α-H), 2.65 (dt,

J = 16.6, 5.1 Hz, 1H, 4-H), 2.54 (s, 3H, NCH3).

13 C NMR (101 MHz, CDCl3) δ/ppm = 165.1 (C-1’’), 158.4 (qPh), 152.2 (C-4’), 151.5 (C-3’’, C- 5’’), 150.0 (C-6), 142.2 (C-7), 136.9 (C-1’), 131.5 (C-2’, C-6’), 131.4 (C-4a), 130.0 (C-8a), 129.6 (Ph), 122.3 (Ph), 121.0 (C-8), 120.5 (C-3’, C-5’), 116.7 (Ph), 112.8 (C-5), 112.1 (C-2’’, C-6’’),

64.6 (C-1), 56.1 (OCH3), 47.5 (C-3), 43.0 (NCH3), 40.0 (C-α), 26.5 (C-4).

-1 IR (ATR) ṽmax/cm = 3406, 2979, 2451, 1636, 1509, 1490, 1273, 1219, 747.

+ HRMS (ESI): calcd. for C29H29N2O3 (M+H) 453.21727; found 453.21781.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

199 EXPERIMENTAL PART

(±)-4-(4-((6-Methoxy-2-methyl-7-phenoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)methyl)- phenoxy)benzonitrile – SG-164 (201)

Following general procedure I, phenol 186 (100 mg, 0.266 mmol, 1.0 eq.), 4-cyanophenyl- boronic acid (117 mg, 0.799 mmol, 3.0 eq.), Cu(OAc)2 (53.2 mg, 0.293 mmol, 1.1 eq.), NEt3

(92.8 µL, 0.666 mmol, 2.5 eq.), pyridine (53.9 µL, 0.666 mmol, 2.5 eq.) and CH2Cl2 (50 mL) were used. After 20 h the reaction was completed and the product was purified by FCC (CH2Cl2

→ 9.7:0.3 CH2Cl2/MeOH) to yield diaryl ether SG-164 (201, 61.1 mg, 0.128 mmol, 48%) as colorless oil. The hydrochloride salt was formed according to general procedure J to yield a colorless solid.

Rf = 0.65 (9:1 CH2Cl2/MeOH). m.p.: 124 °C (HCl salt).

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.56 – 7.51 (m, 2H, 2’’-H, 6’’-H), 7.27 – 7.22 (m, 2H, Ph, collapses with chloroform), 7.10 – 7.06 (m, 2H, 2’-H, 6’-H), 7.01 – 6.96 (m, 1H, Ph), 6.92 – 6.88 (m, 2H, 3’’-H, 5’’-H), 6.88 – 6.81 (m, 4H, 3’-H, 5’-H, Ph), 6.68 (s, 1H, 5-H), 6.37 (s, 1H, 8-

H), 3.77 (s, 3H, OCH3), 3.69 (t, J = 6.1 Hz, 1H, 1-H), 3.18 (ddd, J = 12.8, 7.9, 5.1 Hz, 1H, 3- H), 3.09 (dd, J = 13.8, 5.4 Hz, 1H, α-H), 2.90 – 2.74 (m, 3H, 3-H, 4-H, α-H), 2.65 (dt, J = 16.0,

5.0 Hz, 1H, 4-H), 2.53 (s, 3H, NCH3).

13 C NMR (101 MHz, CDCl3) δ/ppm = 162.0 (C-1’’), 158.4 (qPh), 152.9 (C-4’), 150.0 (C-6), 142.1 (C-7), 136.7 (C-1’), 134.2 (C-2’’, C-6’’), 131.5 (C-2’, C-6’), 131.4 (C-4a), 130.0 (C-8a), 129.6 (Ph), 122.2 (Ph), 121.1 (C-8), 120.2 (C-3’, C-5’), 119.1 (C-4’’), 117.8 (C-3’’, C-5’’), 116.6

(Ph), 112.8 (C-5), 105.6 (CN), 64.6 (C-1), 56.1 (OCH3), 47.4 (C-3), 43.0 (NCH3), 40.0 (C-α), 26.4 (C-4).

-1 IR (ATR) ṽmax/cm = 2940, 2225, 1596, 1495, 1245, 1218, 1166, 836, 750, 736, 690.

+ HRMS (ESI): calcd. for C31H29N2O3 (M+H) 477.21727; found 477.21773.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

200 EXPERIMENTAL PART

(±)-1-(4-(3,4-Dichlorophenoxy)benzyl)-6-methoxy-2-methyl-7-phenoxy-1,2,3,4- tetrahydroisoquinoline – SG-165 (202)

Following general procedure I, phenol 186 (100 mg, 0.266 mmol, 1.0 eq.), 3,4-dichlorophenyl- boronic acid (152 mg, 0.799 mmol, 3.0 eq.), Cu(OAc)2 (53.2 mg, 0.293 mmol, 1.1 eq.), NEt3

(92.8 µL, 0.666 mmol, 2.5 eq.), pyridine (53.9 µL, 0.666 mmol, 2.5 eq.) and CH2Cl2 (20 mL) were used. After 20 h the reaction was completed and the product was purified by FCC (CH2Cl2

→ 9.7:0.3 CH2Cl2/MeOH) to yield diaryl ether SG-165 (202, 109 mg, 0.208 mmol, 78%) as brown oil. The hydrochloride salt was formed according to general procedure J to yield a beige solid.

Rf = 0.58 (9:1 CH2Cl2/MeOH). m.p.: 92 °C (HCl salt).

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.31 (d, J = 8.8 Hz, 1H, 5’’-H), 7.28 – 7.22 (m, 2H, Ph, collapses with chloroform), 7.06 – 6.97 (m, 4H, 2’-H, 6’-H, 2’’-H, Ph), 6.87 – 6.83 (m, 2H, Ph), 6.82 – 6.78 (m, 2H, 3’-H, 5’-H), 6.75 (dd, J = 8.8, 2.8 Hz, 1H, 6’’-H), 6.67 (s, 1H, 5-H), 6.40 (s,

1H, 8-H), 3.78 (s, 3H, OCH3), 3.68 (t, J = 5.6 Hz, 1H, 1-H), 3.17 (ddd, J = 12.6, 7.8, 5.1 Hz, 1H, 3-H), 3.06 (dd, J = 13.9, 5.6 Hz, 1H, α-H), 2.89 – 2.73 (m, 3H, 3-H, 4-H, α-H), 2.64 (dt, J =

15.6, 5.1 Hz, 1H, 4-H), 2.52 (s, 3H, NCH3).

13 C NMR (101 MHz, CDCl3) δ/ppm = 158.3 (qPh), 157.1 (C-1’’ or C-3’’), 154.2 (C-4’), 149.9 (C-6), 142.3 (C-7), 135.8 (C-1’), 133.2 (C-1’’ or C-3’’), 131.3 (C-2’, C-6’), 131.2 (C-4a), 131.0 (C-5’’), 130.1 (C-8a), 129.5 (Ph), 126.1 (C-4’’), 122.3 (Ph), 120.9 (C-8), 120.1 (C-2’’), 119.2 (C-

3’, C-5’), 117.7 (C-6’’), 116.8 (Ph), 112.7 (C-5), 64.6 (C-1), 56.1 (OCH3), 47.6 (C-4), 43.0

(NCH3), 40.1 (C-α), 26.4 (C-4).

-1 IR (ATR) ṽmax/cm = 2979, 1585, 1505, 1489, 1466, 1261, 1223, 1119, 750.

35 + HRMS (ESI): calcd. for C30H28 Cl2NO3 (M+H) 520.14408; found 520.14478.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

201 EXPERIMENTAL PART

5.2.2.6 Further reactions

(±)-7-(benzyloxy)-1-(4-(benzyloxy)-3-methoxybenzyl)-2-ethyl-6-methoxy-1,2,3,4- tetrahydroisoquinoline – SG-158 (208)

(±)-7-(Benzyloxy)-1-(4-(benzyloxy)-3-methoxybenzyl)-6-methoxy-1,2,3,4- tetrahydroisoquinoline (Z6 (145), 100 mg, 0.188 mmol, 1.0 eq.), NEt3 (65.5 µL, 0.470 mmol,

2.5 eq.) and KI (31.2 mg, 0.188 mmol, 1.0 eq.) were dispersed in CHCl3 (20 mL) and bromoethane (30.8 µL, 0.282 mmol, 1.5 eq.) was added. The reaction mixture was stirred at

65 °C for 6 days and subsequently concentrated in vacuo. The residue was dissolved in CH2Cl2

(30 mL) and washed with sat. aq. NaHCO3 solution (10 mL), 10% aq. citric acid solution

(10 mL) and sat. aq. NaCl solution (10 mL). The organic layer was dried over Na2SO4, filtered and concentrated in vacuo. Purification by FCC (CH2Cl2 → 9.7:0.3 CH2Cl2/MeOH), followed by purification via preparative HPLC (6:4 n-heptane/isopropanol + 0.45% diethylamine, 15 mL flow) yielded racemic amine SG-158 (208, 23.2 mg, 0.0443 mmol, 24%) as colorless oil. The hydrochloride salt was formed according to general procedure J to yield a colorless solid.

Rf = 0.54 (9:1 CH2Cl2/MeOH). m.p.: 211 °C (HCl salt).

1 H NMR (500 MHz, CDCl3) δ/ppm = 7.45 – 7.27 (m, 10H, PhCH2, collapses with chloroform), 6.80 (d, J = 8.2 Hz, 1H, 5’-H), 6.62 (d, J = 1.9 Hz, 1H, 2’-H), 6.59 (s, 1H, 5-H), 6.55 (dd, J =

8.3, 2.0 Hz, 1H, 6’-H), 6.05 (s, 1H, 8-H), 5.13 (s, 2H, PhCH2), 4.85 (d, J = 12.3 Hz, 1H, PhCH2),

4.74 (d, J = 12.3 Hz, 1H, PhCH2), 3.86 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.75 (dd, J = 7.4, 6.0 Hz, 1H, 1-H), 3.21 – 3.13 (m, 1H, 4-H), 3.04 (dd, J = 13.5, 5.2 Hz, 1H, α-H), 2.91 – 2.81

(m, 2H, 3-H, 4-H), 2.72 – 2.66 (m, 3H, α-H, CH2CH3), 2.55 – 2.47 (m, 1H, 3-H), 1.12 (t, J = 7.1

Hz, 3H, CH2CH3).

13 C NMR (126 MHz, CDCl3) δ/ppm = 149.4 (C-3’), 148.0 (C-6), 146.6 (C-4’), 145.6 (C-7), 137.5

(qPhCH2), 137.5 (qPhCH2), 133.6 (C-1’), 129.8 (C-8a), 128.6 (PhCH2), 128.5 (PhCH2), 127.9

(PhCH2), 127.8 (PhCH2), 127.4 (PhCH2), 127.3 (PhCH2), 127.1 (C-4a), 122.1 (C-6’), 114.2 (C-

2’), 113.9 (C-8 or C-5’), 113.8 (C-8 or C-5’), 111.9 (C-5), 71.3 (PhCH2), 71.0 (PhCH2), 62.6 (C-

1), 56.1 (2 OCH3), 47.8 (CH2CH3), 43.6 (C-3), 40.8 (C-α), 25.5 (C-4), 13.4 (CH2CH3).

-1 IR (ATR) ṽmax/cm = 2998, 2359, 1589, 1514, 1264, 1226, 1021, 737, 696.

202 EXPERIMENTAL PART

+ HRMS (ESI): calcd. for C34H38NO4 (M+H) 524.27954; found 524.28026.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

(±)-7-(Benzyloxy)-1-(4-(benzyloxy)-3-methoxybenzyl)-6-methoxy-2-methyl-1,2,3,4- tetrahydroisoquinoline – SG-159 (206)

A solution of (±)-7-(benzyloxy)-1-(4-(benzyloxy)-3-methoxybenzyl)-6-methoxy-1,2,3,4- tetrahydro-isoquinoline (Z6 (145),100 mg, 0.188 mmol, 1.0 eq.) and 37% aqueous formaldehyde (70.0 µL, 0.940 mmol, 5.0 eq.) in HPLC grade MeOH (5.0 mL) was acidified with glacial acetic acid (0.50 mL, final pH ~ 5), before NaCNBH3 (24.9 mg, 0.376 mmol, 2.0 eq.) was added. The reaction mixture was stirred for 2 days at rt and concentrated in vacuo to give a light yellow solid. The residue was dissolved in EtOAc (40 mL) and was washed with sat. aq.

NaHCO3 solution (15 mL), 10% aq. citric acid solution (15 mL) and sat. aq. NaCl solution

(15 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The crude product was purified by FCC (CH2Cl2 → 9.7:0.3 CH2Cl2/MeOH) to yield racemic amine SG-159 (206, 61.2 mg, 0.120 mmol, 64%) as colorless oil. The hydrochloride salt was formed according to general procedure J to yield a colorless solid.

Rf = 0.57 (9:1 CH2Cl2/MeOH). m.p.: 87 °C (HCl salt).

1 H NMR (400 MHz, CDCl3) δ/ppm = 7.42 – 7.23 (m, 13H, PhCH2, collapses with chloroform), 6.76 (d, J = 8.1 Hz, 1H, 5’-H), 6.61 – 6.54 (m, 2H, 5-H, 2’-H), 6.51 (dd, J = 8.1, 1.9 Hz, 1H, 6’-

H), 6.08 (s, 1H, 8-H), 5.11 (s, 2H, PhCH2), 4.84 (d, J = 12.3 Hz, 1H, PhCH2), 4.72 (d, J = 12.3

Hz, 1H, PhCH2), 3.84 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.61 (dd, J = 7.2, 5.0 Hz, 1H, 1-H), 3.17 – 3.02 (m, 2H, 3-H, α-H), 2.84 – 2.67 (m, 3H, 3-H, 4-H, α-H), 2.60 – 2.53 (m, 1H, 4-H),

2.51 (s, 3H, NCH3).

13 C NMR (101 MHz, CDCl3) δ/ppm = 149.3 (C-3’), 147.9 (C-6), 146.6 (C-4’), 145.6 (C-7), 137.5

(qPhCH2), 137.4 (qPhCH2), 133.2 (C-1’), 129.3 (C-8a), 128.7 (PhCH2), 128.6 (PhCH2), 127.9

(PhCH2), 127.8 (PhCH2), 127.4 (PhCH2), 127.3 (PhCH2), 126.8 (C-4a), 122.0 (C-6’), 113.8 (C-

203 EXPERIMENTAL PART

8 or C-5’), 113.7 (C-8 or C-5’), 113.5 (C-2’), 111.7 (C-5), 71.2 (PhCH2), 70.9 (PhCH2), 64.8 (C-

1), 56.1 (2 OCH3), 47.2 (C-3), 42.9 (NCH3), 40.8 (C-α), 25.9 (C-4).

-1 IR (ATR) ṽmax/cm = 2980, 2889, 2359, 1513, 1263, 1225, 1012, 696.

+ HRMS (ESI): calcd. for C33H36NO4 (M+H) 510.26389; found 510.26339.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

(±)-7-(Benzyloxy)-1-(4-(benzyloxy)benzyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydro- isoquinoline – SG-005 (187)

An alternative route to receive SG-005 (187): A solution of Z3 (137, 251 mg, 0.500 mmol, 1.0 eq.) and 37% aq. formaldehyde solution (68.9 µL, 2.50 mmol, 5.0 eq.) in HPLC grade MeOH (5.0 mL) was acidified with two drops of glacial acetic acid (0.50 mL, final pH ~ 5) and sodium cyanoborohydride (66.1 mg, 1.00 mmol, 2.0 eq.) was added. The reaction mixture was stirred for 4 days at rt and concentrated in vacuo to give a white solid. The residue was dissolved in

EtOAc (40 mL) and was washed with sat. aq. NaHCO3 solution (15 mL), 10% aq. citric acid solution (15 mL) and sat. aq. NaCl solution (15 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The crude product was purified by FCC (9.5:0.5 CHCl3/MeOH) to yield SG-005 (187, 133 mg, 0.278 mmol, 56%) as off white solid. Analytical data are stated above.

(±)-7-(Benzyloxy)-1-(4-(benzyloxy)benzyl)-6-methoxy-2-methyl-1,2,3,4-tetrahydro- isoquinoline – SG-005 (187)

An alternative route to receive SG-005 (187) via Mitsunobu reaction: SG-132 (159, 150 mg,

0.500 mmol, 1.0 eq.) is dissolved in dry THF (5.0 mL), before PPh3 (629 mg, 2.40 mmol, 4.8 eq.) and benzyl alcohol (145 µL, 1.40 mmol, 2.8 eq.) were added. The reaction mixture was cooled to 4 °C and DIAD (315 µL, 1.60 mmol, 3.2 eq.) was added dropwise. The mixture was allowed to warm to rt for 18 h and was then concentrated in vacuo. The residue was purified by FCC (9.9:0.1 → 9.7:0.3 CH2Cl2/MeOH) to yield SG-005 (187, 113 mg, 0.236 mmol, 47%) as light yellow oil. Analytical data are stated above.

5.2.2.7 Substances received from other sources

Prof. Dr. Peter Pachaly† kindly provided tetrandrine (1) and fangchinoline (9) from natural sources and semi synthetic N,N-dimethyl-tetrandrine dichloride (139, Table 15). Prof. Dr.

204 EXPERIMENTAL PART

Meinhart Zenk† kindly provided a compound library of 1-benzyl-1,2,3,4-tetrahydroisoquinolines (Z compounds, Table 15).

Table 15: Substances received from other sources. *Analytical data are stated below.

entry name abbreviation literature

1 tetrandrine (1) - [143]

2 fangchinoline (9) - [144]

3 N,N- dimethyltetrandrine dichloride (139) - [145]

4 (±)-coclaurine hydrochloride (141) Z1 [194, 195]

5 R-coclaurine hydrochloride (142) Z2 [194, 195]

(±)-7-(benzyloxy)-1-(4-(benzyloxy)benzyl)-6-methoxy-1,2,3,4- 6 Z3 [82, 195]* tetrahydroisoquinoline hydrochloride (137)

(+)-O,O-dibenzyl coclaurine / (+)-7-(benzyloxy)-1-(4- 7 (benzyloxy)benzyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline Z4 [193] (143)

(±)-6-(benzyloxy)-1-(4-(benzyloxy)benzyl)-7-methoxy-1,2,3,4- 8 Z5 [196] tetrahydroisoquinoline hydrochloride (144)

(±)-O,O-dibenzyl nororientaline / (±)-7-(benzyloxy)-1-(4- 9 (benzyloxy)-3-methoxy-benzyl)-6-methoxy-1,2,3,4- Z6 [197] tetrahydroisoquinoline (145)

10 R-norreticuline (146) Z7/Z8 [198, 199]

11 (±)-laudanosine (147) Z9 [200]

(±)-6-(benzyloxy)-1-(3-(benzyloxy)-4-methoxybenzyl)-7- 12 Z11 [201] methoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (148)

(±)-6,7-bis(benzyloxy)-1-(4-(benzyloxy)-3-methoxybenzyl)-2- 13 Z12 [202] methyl-1,2,3,4-tetrahydroisoquinoline hydrochloride (149)

(±)-5-(3,4-bis(benzyloxy)benzyl)-5,6,7,8-tetrahydro- 14 Z13 * [1,3]dioxolo[4,5-g]isoquinoline hydrochloride (150)

(±)-1-(benzo[d][1,3]dioxol-5-ylmethyl)-6,7-dimethoxy-1,2,3,4- 15 Z14 [203, 204] tetrahydroisoquinoline (151)

(±)-1-(benzo[d][1,3]dioxol-5-ylmethyl)-6-(benzyloxy)-7-methoxy- 16 Z15 [205] 1,2,3,4-tetrahydroisoquinoline (152)

(±)-1-(benzo[d][1,3]dioxol-5-ylmethyl)-6,7-bis(benzyloxy)- 17 Z16/Z17 [206] 1,2,3,4-tetrahydroisoquin-oline (153)

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(±)-1-(benzo[d][1,3]dioxol-5-ylmethyl)-6,7-bis(benzyloxy)-3,4- 18 Z18 [206] dihydroisoquinoline (154)

(±)-6,7-bis(benzyloxy)-1-((6-bromobenzo[d][1,3]dioxol-5- 19 Z20 * yl)methyl)-1,2,3,4-tetrahydro-isoquinoline hydrochloride (155)

(±)-1-((6-bromobenzo[d][1,3]dioxol-5-yl)methyl)-3,4- 20 Z21 [207] dihydroisoquinoline-6,7-diol (156)

N-(4-(benzyloxy)-3-methoxyphenethyl)-2-(4-(benzyloxy)- 21 Z22 [208] phenyl)acetamide (157)

Epiberberine chloride / 8,9-dimethoxy-11,12-dihydro- 22 [1,3]dioxolo-[4,5-h]isoquinolino[2,1-b]isoquinolin-13-ium chloride Z23 [209] (158)

(±)-7-(benzyloxy)-1-(4-(benzyloxy)benzyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride – Z3 (137)

1 H NMR (500 MHz, CD3OD) δ/ppm = 7.51 – 7.25 (m, 10H), 7.21 – 7.12 (m, 2H), 7.08 – 6.99 (m, 2H), 6.84 (s, 1H), 6.45 (s, 1H), 5.09 (s, 2H), 4.84 (d, J = 2.7 Hz, 2H), 4.64 (t, J = 7.3 Hz, 1H), 3.85 (s, 3H), 3.61 – 3.47 (m, 1H), 3.42 – 3.34 (m, 1H), 3.31 – 3.24 (m, 1H), 3.17 – 3.00 (m, 3H).

13 C NMR (126 MHz, CD3OD) δ/ppm = 159.8, 151.2, 148.0, 138.6, 138.3, 132.0, 129.5, 129.5, 129.0, 128.9, 128.9, 128.7, 128.6, 125.5, 124.3, 116.5, 114.0, 113.2, 72.0, 71.0, 57.4, 56.5, 49.5, 49.3, 49.2, 49.0, 48.8, 48.7, 48.5, 40.3, 40.1, 25.8.

+ HRMS (ESI): calcd. for C31H32NO3 (M+H) 466.23767; found 466.23798.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

(±)-O,O-dibenzyl nororientaline / (±)-7-(benzyloxy)-1-(4-(benzyloxy)-3-methoxy-benzyl)- 6-methoxy-1,2,3,4-tetrahydroisoquinoline – Z6 (145)

1 H NMR (500 MHz, CDCl3) δ/ppm = 9.27 (s, 1H), 7.51 – 7.23 (m, 10H), 7.04 – 6.99 (m, 2H), 6.81 (s, 1H), 6.79 (dd, J = 8.2, 1.9 Hz, 1H), 6.63 (s, 1H), 5.05 (s, 2H), 4.90 – 4.76 (m, 2H), 4.59 (t, J = 7.0 Hz, 1H), 3.75 (s, 6H), 3.32 – 3.29 (m, 1H), 3.27 – 3.09 (m, 3H), 3.03 – 2.87 (m, 2H).

13 C NMR (126 MHz, CDCl3) δ/ppm = 159.8, 151.2, 148.0, 138.6, 138.3, 132.0, 129.6, 129.5, 129.0, 128.9, 128.8, 128.7, 128.6, 125.5, 124.3, 116.5, 114.0, 113.2, 72.0, 71.0, 57.4, 56.5, 40.3, 40.1, 25.8.

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+ HRMS (ESI): calcd. for C32H34NO4 (M+H) 496.24824; found 496.24827.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

(±)-5-(3,4-bis(benzyloxy)benzyl)-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinoline hydrochloride – Z13 (150)

1 H NMR (500 MHz, CDCl3) δ/ppm = 10.22 (d, J = 7.6 Hz, 1H), 9.66 (d, J = 8.9 Hz, 1H), 7.42 – 7.24 (m, 10H), 6.88 – 6.84 (m, 1H), 6.82 (s, 1H), 6.73 (dd, J = 8.2, 1.9 Hz, 1H), 6.51 (s, 1H), 6.26 (s, 1H), 5.92 (d, J = 1.3 Hz, 1H), 5.89 (d, J = 1.2 Hz, 1H), 5.16 – 5.00 (m, 4H), 4.68 – 4.57 (m, 1H), 3.44 – 3.34 (m, 1H), 3.27 – 3.16 (m, 2H), 3.08 – 2.95 (m, 2H), 2.83 – 2.71 (m, 1H).

13 C NMR (126 MHz, CDCl3) δ/ppm = 149.1, 148.4, 147.6, 146.8, 137.4, 128.7, 128.6, 128.4, 127.9, 127.6, 127.5, 125.5, 124.2, 123.1, 121.5, 116.8, 115.6, 108.8, 107.0, 101.4, 77.4, 76.9, 71.4, 55.3, 40.5, 38.9, 25.6.

+ HRMS (ESI): calcd. for C31H30NO4 (M+H) 480.21693; found 480.21719.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

(±)-6,7-bis(benzyloxy)-1-((6-bromobenzo[d][1,3]dioxol-5-yl)methyl)-1,2,3,4-tetrahydro- isoquinoline hydrochloride – Z20 (155)

1 H NMR (500 MHz, (CD3)2SO) δ/ppm = 9.30 (s, 1H), 7.51 – 7.24 (m, 10H), 7.18 (s, 1H), 6.96 (s, 1H), 6.60 (s, 1H), 6.08 (d, J = 7.7 Hz, 2H), 5.13 (s, 2H), 5.07 – 4.92 (m, 2H), 4.61 – 4.41 (m, 1H), 3.47 – 3.37 (m, 1H), 3.37 – 3.25 (m, 3H), 3.25 – 3.14 (m, 2H), 3.12 – 3.00 (m, 1H), 2.91 – 2.77 (m, 1H).

13 C NMR (126 MHz, (CD3)2SO) δ/ppm = 147.8, 147.7, 147.3, 146.5, 137.0, 128.4, 128.4, 128.2, 127.8, 127.8, 127.6, 127.3, 125.0, 123.8, 115.0, 114.1, 112.7, 112.5, 111.9, 102.1, 70.2, 70.0, 53.6, 38.8, 24.5.

79 + HRMS (ESI): calcd. for C31H29 BrNO4 (M+H) 558.12745; found 558.12829.

Purity (HPLC): > 96% (λ = 210 nm), > 96% (λ = 254 nm).

Berbamine dihydrochloride (10) was purchased from Sigma-Aldrich (now Merck, Darmstadt, Germany), cepharanthine (140) and dauricine (11) from Carbosynth (Compton, Berkshire, United Kingdom) and oxyacanthine sulfate (138) from abcr (Karlsruhe, Germany).

207 EXPERIMENTAL PART

5.3 Biological methods 5.3.1 High-throughput screening (HTS)

The high-throughput screening was performed by Dr. Phuong Nguyen and myself in collaboration with the group of Prof. Dr. Michael Schaefer at the Rudolf-Böhm-Institut für Pharmakologie und Toxikologie, University of Leipzig. The experiments were run using a custom-made fluorescence imaging plate reader built into a robotic liquid handling station (Freedom Evo 150, Tecan, Männedorf, Switzerland) as previously described[22, 88].

In brief, HEK-293 cells stably expressing human TPC2L11A/L12A-RFP and stably expressing

L253A/I254A human CLN3 -RFP were cultured at 37 °C with 5% of CO2 in Dulbecco’s modified Eagle medium (Thermo Fisher), supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany), 2.00 mM L-glutamine, 100 U/mL penicillin, 0.100 mg/mL streptomycin, and 400- 800 µg/mL G418. Cells were incubated with Fluo-4/AM (15, 4.00 µM; Life Technologies, Eugene, Oregon, USA) for 30 min at 37 °C, washed and resuspended in a HEPES-buffered solution 1 (HBS1) comprising 132 mM NaCl, 6.00 mM KCl, 1.00 mM MgCl2, 1.00 mM CaCl2, 10.0 mM HEPES, and 5.50 mM D-glucose (pH was adjusted to 7.4 with NaOH). Then, the cells were seeded on black walled, clear bottom 384-well plates (Greiner, Frickenhausen, Germany) and the plates were placed in the imaging reader. Experiments were performed with cell suspensions.

For primary screening, individual compounds from Roche libraries (Xplore libraries X30 and X50, Roche, Basel, Switzerland) were diluted in HBS1 to a working concentration of 100 µM. After recording the baseline for 30 s, compounds were injected to a final concentration of 10 µM. Recording continued for 180 s per quadrant (total 750 s for four quadrants). If high intensities were measured in both cell lines, the compounds were deemed false positives and excluded. From single hits with high intensities concentration effect experiments were performed. In case of a low IC50, hits were confirmed by single cell Ca-imaging. Concentration effect relationships were plotted using GraphPad Prism 5 and fitted to the Hill equation.

Concentration-effect experiments were performed by Nicole Urban (Schäfer group) as described above.

5.3.2 Single cell calcium imaging

Single cell Ca2+ imaging experiments were performed as previously described[22, 115]. In more detail, HEK293 cells were cultured at 37 °C with 5% of CO2 in Dulbecco’s modified Eagle medium (Gibco), supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 0.100 mg/mL streptomycin. Cells were plated onto poly-L-lysine (sigma)-coated glass coverslips, grown over two days and transiently transfected for 18-24 h with plasmids using TurboFect

208 EXPERIMENTAL PART

(Thermo Fisher Scientific, Waltham, USA) according to the manufacturer’s instructions. Plasmids used for transfection were generated in the group of Prof. Christian Grimm and are literature known. Experiments for blockers were performed using HEK293 cells stably expressing TPC2L11A/L12A –RFP[14, 22, 28, 114, 115].

Transfected cells were loaded for 1 h at 37 °C with 10% of CO2 with Fura-2/AM (126, 4.0 µM) and 0.005% (v/v) Pluronic® F-127 (both from Thermo Fisher) in HEPES-buffered solution 2

(HBS2) comprising 138 mM NaCl, 6.00 mM KCl, 1.00 mM MgCl2, 2.00 mM CaCl2, 10.0 mM HEPES, and 5.50 mM D-glucose (pH was adjusted to 7.4 with NaOH). After loading, cells were washed in HBS2, and mounted in an imaging chamber. All recordings were performed in HBS2. Images were acquired every 2 s at 40X magnification using a monochromator-based imaging system (Polychrome IV mono-chromator, TILL photonics or a Leica DMi8 live cell microscope). Fura-2 (13) was excited at 340 nm/380 nm and emitted fluorescence was captured using 515 nm long-pass filters.

Compound stock solutions (10 mM in DMSO) were pre diluted with HBS2 to a working concentration of 100 µM (1% DMSO). Chambers were filled with 450 µL HBS2 and the baseline was recorded for 50 s. Then, 50 µL of the compound solution to be tested were added to reach a final concentration of 10 µM. After 400 s the experiments were stopped. If no activation was observed or an inhibitor should be tested, a known activator was added to confirm the correctness of the experiment or if the substance is a blocker. Activation/inhibition was illustrated using GraphPad Prism 5.

5.3.3 MTT

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed by Martina Stadler (Bracher group) and conducted with HL-60 cells. First the number of cells per mL was determined with a hepatocyte cell counter (Fuchs-Rosenthal). Then the cell suspension was diluted with medium to 9 x 105 cells mL-1.

Compounds to be tested were dissolved in DMSO to give 10 mM stock solutions and used for a dilution series (10 mM, 5 mM, 2.5 mM, 1.25 mM, 0.625 mM, 0.3125 mM). For negative control 1% DMSO was used and for positive control Triton® X-100 solution with a final concentration of 1 µg/mL was added.

Cell suspensions (99 µL each) were seeded on 96-well plates and incubated at 37 °C with 5%

CO2 for 24 h. Then, compound solutions (1 µL) were added and again incubated at 37 °C with

5% CO2 for 24 h. After that, 10 µL MTT solution (5.0 mg MTT in 1.0 mL PBS) was added to each well and further incubated for two hours, followed by addition of 190 µL DMSO. After one hour shaking continuously, photometric quantification was conducted at a wavelength of 570

209 EXPERIMENTAL PART nm with an MRX microplate reader (DYNEX Technologies, Chentilly, USA). Concentration- effect relationships were plotted using GraphPad Prism 5.

5.3.4 Agar diffusion test

Agar diffusion tests were performed by Martina Stadler (Bracher group). Microorganisms were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig) and cultivated according to recommendations in liquid culture using different agars. For Saccharomyces cerevisiae (DSM number: 1333), Hyphopichia burtonii (DSM number: 70663), Yarrowia lipolytica (DSM number: 1345), Escherichia coli (DSM number: 426) and Pseudomonas marginalis (DSM number: 7527) all-culture agar (AC-agar, Sigma Aldrich) was used. Therefore 35.2 g AC-agar and 20 g agar were suspended in 1.0 L water and treated by autoclave. For Staphylococcus equorum (DSM number: 20675) and Streptococcus entericus (DSM number: 14446) an agar is likewise prepared from 10.0 g caseinpeptone, 5.0 g yeast extract, 5.0 g glucose and 5.0 g sodium chloride in 1.0 L water. After treatment in the autoclave 15 mL of the warm, liquid agar was filled into Petri dishes and cooled to 8 °C for one hour.

Solutions with 1% (m/V) compound in DMSO were prepared. Then, 3.0 µL of each solution was plated onto small filter plates (diameter 6 mm, Macherey-Nagel), equivalent to 30 µg substance. As positive control clotrimazole and tetracycline were used. Blind control was conducted with mere DMSO. The small filter plates were then dried for 24 hours at room temperature.

For final experiments the germs were brought onto the different agars using cotton swabs. The platelets containing the substances, the reference, and the blind control were put onto the agar, too. The agar plates were incubated for 36 h at 32 °C (bacteria) or 28 °C (yeasts). Then, the diameters of growth inhibition (inhibition zones) were measured manually.

210 APPENDICES

6 Appendices 6.1 Abbreviations

5‐HT 5‐hydroxytryptamine receptors Å Angstrom ALS amyotrophic lateral sclerosis AM acetoxymethyl APCI atmospheric-pressure chemical ionization aq. aqueous ASAP atmospheric pressure solids analysis probe BK large conductance calcium-activated potassium channels/big potassium Bn benzyl calcd. calculated Cav voltage gated calcium channel CB1R cannabinoid‐1 receptor CLN3 battenin CTB cell titer blue cy cyclohexyl CYP cytochrome P DCC N,N′-dicyclohexylcarbodiimide DIPA diisopropylamine DMF dimethylformamide DMSO dimethyl sulfoxide ECD Electronic circular dichroism spectra EDC ethylene dichloride EGF epidermal growth factor EI electron ionization eq. equivalents ESI electron spray ionization FCC flash column chromatography h hour HCT-15 human colorectal adenocarcinoma cells HEK293 human embryonic kidney cells HepaRG hepatic stem cells HepG2 human hepatocellular carcinoma cells HERG human Ether-à-go-go-Related Gene HIV human immunodeficiency virus HPLC high-performance liquid chromatography HRMS high-resolution mass spectrometry HSQC heteronuclear single quantum correlation HTS high-throughput screening HUVEC human umbilical vein endothelial cells

211 APPENDICES

Hz hertz IR infrared spectroscopy JNCL juvenile neuronal ceroid lipofuscinosis KATP ATP-sensitive potassium channel LDA lithium diisopropylamide LHMDS lithium bis(trimethylsilyl)amide LOPAC Library of Pharmacologically Active Compounds LRRK2 leucine‐rich repeat kinase 2 LSD Lysosomal storage disease m meta m multiplet (NMR) M molar m.p. melting point Me methyl MERS‐CoV Middle East respiratory syndrome coronavirus min minutes mmol millimole mol mole MS multiple sclerosis mTOR mechanistic target of rapamycin MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid mw microwave n.a. not applicable NAADP nicotinic acid adenine dinucleotide phosphate Nav voltage gated sodium channel NBS N-bromosuccinimide n-BuLi n-butyllithium NMDA N ‐methyl‐D‐aspartate receptor NMR nuclear magnetic resonance ns not significant NT non-transfected o ortho p para PBMC peripheral blood mononuclear cells PG protecting group P-gp P-glycoprotein ph phenyl

PI(3,5)P2 phosphatidylinositol 3,5‐bisphosphate PIKfyve FYVE finger-containing phosphoinositide kinase ppm parts per million q quartet (NMR)

Rf retardation factor

212 APPENDICES

RFP red fluorescent protein rt room temperature s singlet (NMR) SAR structure-activity relationships SARS-CoV-2 severe acute respiratory syndrome coronavirus 2 sat. saturated SEM standard error of the mean SERM selective estrogen receptor modulators SSRI selective serotonin reuptake inhibitors TCA tricyclic antidepressants TFA trifluoroacetic acid TFEB transcription factor EB THF tetrahydrofuran TLC thin-layer chromatography TPC two pore channel TRPA transient receptor potential channel ankyrin TRPC transient receptor potential channel canonical TRPM transient receptor potential channel melastatin TRPML transient receptor potential cation channel TRPV transient receptor potential channel vanilloid TTX tetrodotoxin VCR-R CEM vincristine-resistant acute lymphoblastic leukemia cells VEGF vascular endothelial growth factors WHO world health organization YFP yellow fluorescent protein

213 APPENDICES

6.2 References

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